Compositions and methods of altering cholesterol levels

ABSTRACT

The present invention relates to compositions, methods and kits using polynucleotides, primary transcripts and mmRNA molecules.

REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/454,977, filed Aug. 8, 2014, which is a continuation of U.S. patentapplication Ser. No. 14/135,887, filed Dec. 20, 2013, now U.S. Pat. No.8,980,864, entitled Compositions and Methods of Altering CholesterolLevels, which claims priority to U.S. Provisional Patent Application No.61/786,737, filed Mar. 15, 2013, entitled Compositions and Methods ofAltering Cholesterol Levels; U.S. Provisional Patent Application No.61/828,214, filed May 29, 2013, entitled Compositions and Methods ofAltering Cholesterol Levels; U.S. Provisional Patent Application No.61/839,488, filed Jun. 26, 2013, entitled Compositions and Methods ofAltering Cholesterol Levels; U.S. Provisional Patent Application No.61/903,474, filed Nov. 13, 2013, entitled Compositions and Methods ofAltering Cholesterol Levels, the contents of each of which is hereinincorporated by reference in its entirety.

REFERENCE TO SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a filed entitled“3529_0190007_SeqListing.txt,” created on Mar. 8, 2017, which is 175,575bytes in size. The information in the electronic format of the sequencelisting is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention is directed to compositions and methods formodulating and/or altering cholesterol levels in an organism or alteringcholesterol trafficking in an organism. In one aspect, the inventionrelates to modified RNA in therapeutics. The modified RNA of theinvention may encode peptides, polypeptides or multiple proteins. Themodified RNA of the invention may also be used to produce polypeptidesof interest. The modified RNA molecules of the invention may thereforebe referred to as modified mRNA. The polypeptides of interest may beused in therapeutics and/or clinical and research settings.

BACKGROUND OF THE INVENTION

High cholesterol is one of a number of risk factors for heart attack andstroke. Although poor diet and lack of excise are common causes of highcholesterolgenetic changes, such as familiar hypercholesterolemia (FH),which is caused by deficiency in LDLR, can be causes of highcholesterol. A number of cholesterol lowering drugs are currently on themarket but they are not without risk or contraindications with certainconditions or other medications. Such drugs include statins, fibrates,niacin, bile acid sequestrants (resins), phytosterols, or othercompounds that prevent absorption of fats, reduce absorption ofcholesterol, or target genes in the cholesterol trafficking pathway.

Nucleic acid based cholesterol lowering drugs include, for example anantisense oligonucleotide inhibitor which targets ApoB-100, mipomersen,which was approved in January 2013 for the treatment of homozygousfamilial hypercholesterolemia (FH). In December of 2012, the FDA alsoapproved lomitapide for the same condition.

More troubling are the liver related problems associated withcholesterol targeting drugs, particularly elevation in serumtransaminases and accumulation of hepatic fat (or hepatic steatosis).For example, because of the potentially significant safety concernssurrounding mipomersen, the drug will carry a boxed warning about livertoxicity as well as requiring certification of prescribers andpharmacies, as well as documentation that the drug is being properlyused with each new prescription. While mipomersen was generallyeffective in lowering LDL cholesterol (more than half of patients inclinical trials had more than a 20% decrease in LDL levels and in thehomozygous FH trial, it reduced LDL by 24.7%), a typical FH patient hasan average LDL between 400-1000 mg/dL. Consequently, lowering was notlikely enough in these patients. In addition, the trials were not largeenough to be powered to assess cardiovascular outcomes, thoughcardiovascular benefit is of course the ultimate intended effect of thedrug. Further, serious adverse events of cardiac disorders occurred inthe mipomersen group in phase 3 trials.

The present invention addresses both the problem of elevated LDLcholesterol levels and dysregulation of hepatic function by providingnucleic acid based compounds or polynucleotides which encode apolypeptide of interest (e.g., modified mRNA or mmRNA) and which havestructural and/or chemical features that avoid one or more of theproblems in the art.

To this end, the inventors have shown that certain modified mRNAsequences have the potential as therapeutics with benefits beyond justevading, avoiding or diminishing the immune response. Such studies aredetailed in published co-pending applications International ApplicationPCT/US2011/046861 filed Aug. 5, 2011 and PCT/US2011/054636 filed Oct. 3,2011, International Application number PCT/US2011/054617 filed Oct. 3,2011, the contents of which are incorporated herein by reference intheir entirety.

SUMMARY OF THE INVENTION

Described herein are compositions, methods and kits RNA using modifiedRNA in the treatment, prevention or diagnosis of disease and disordersassociated with cholesterol and/or cholesterol trafficking.

According to the present invention, the pathways associated withcholesterol trafficking are modulated by providing one or morepolypeptides (including enzymes) which alter either the concentrationsof cholesterol, its processing or transport.

In one embodiment, the transport of LDL cholesterol from plasma to livercells is increased by providing the cell with either more receptormolecules or by minimizing the destruction of the LDL receptor. In thefirst instance a polynucleotide, primary construct or mmRNA is providedwhich encodes LDL receptor. In the second instance, a mutant form of LDLreceptor is encoded by the polynucleotide, primary construct or mmRNA.Such mutant LDL receptors (LDL-R or LDLR) would be deficient in some wayin their binding of PCSK-9. Accordingly, a PCSK9 binding deficient LDLRwould bring cholesterol into the hepatocyte.

Provided herein are polynucleotides, primary constructs and/or mmRNAwhich encode an LDLR mutant. In one aspect, a modified mRNA may encode aLDLR mutant which may comprise at least one amino acid mutation in aregion comprising amino acids 314-393 of LDLR such as, but not limitedto, SEQ ID NO: 19. In one embodiment, the region of amino acidscomprises amino acids 316-339 of SEQ ID NO: 19. The modified mRNA maycomprise at least one nucleoside modification such as, but not limitedto, 1-methylpseudouridine. The modified mRNA may also comprise thenucleoside modification 5-methylcytosine.

Provided herein are also methods of reducing serum cholesterol in asubject comprising administering to the subject a modified mRNA mayencode a LDLR mutant which may comprise at least one amino acid mutationin a region comprising amino acids 314-393 of LDLR such as, but notlimited to, SEQ ID NO: 19. In one embodiment, the region of amino acidscomprises amino acids 316-339 of SEQ ID NO: 19.

In one embodiment, the hepatocyte is provided with one or morepolynucleotides, primary constructs, or mmRNA encoding and/or whichoverexpresses CYP7A1. CYP7A1 is the rate limiting enzyme for bile acidsynthesis, and promotes removal of the incoming cholesterol. There arehumans with CYP7A1 mutations that are associated with high plasmalow-density lipoprotein (LDL) and hepatic cholesterol content, as wellas deficient bile acid excretion.

In one embodiment, two polynucleotides, primary constructs, or mmRNA aredelivered resulting in lower plasma cholesterol and concomitant enhancedcholesterol disposal.

In one embodiment, the one or more polynucleotides, primary constructsor mmRNA are modified in the 3′UTR to contain a microRNA binding site orseed. In this embodiment, the CYP7A1polynucleotide, having a normal halflife of approximately 30 minutes, may be made transcription-dependent bymiR-destabilization (specifically the ubiquitous miR122a inhepatocytes). According to this embodiment, a miR122a binding site maybe incorporated into the 3′UTR of the mmRNA rendering the transcriptless stable. This would allow, depending on the number of binding sitesengineered into the construct, the titration of stability and thereforeallow for control of expression of the encoded CYP7A1 enzyme. Thepolynucleotides, primary constructs primary constructs or mmRNA encodingCYP7A1 may also be useful in creating mouse models useful in proof ofconcept studies and basic research. These studies would be analogous toproducing dose dependent gene therapy.

In one embodiment, treatment regimes may be designed for rare diseaseswhere patients present with CYP7A1 polymorphisms that are hyporesponsiveto statins. In this instance, studies of effective compositions of thepresent invention may be completed quickly and based on diet-basedchallenges. As such the compositions of the present invention are usefulin the study and treatment of disease involving cholesterol relateddiseases, both rare and prevalent.

In one embodiment, the compositions of the present invention may beadministered along with other drug compounds. Such other drugs includespecifically statins. Examples of statins include, but are not limitedto, atorvastatin, cerivastatin, fluvastatin, lovastatin, mevastatin,pitavastatin, pravastatin, simvastatin, and combinations thereof.

According to the present invention, the compositions comprisingpolynucleotides, primary constructs or mmRNA are useful in treatingdiseases such as rare non-alcoholic fatty liver disease. In treatingthis disorder, it is contemplated that any therapeutic that driveshepatic cholesterol to its natural sink (out of the body through biles)would have superior treatment outcomes. Consequently, it is contemplatedthat administration of polynucleotides, primary constructs or mmRNAencoding LDLR would increase cholesterol in the hepatocyte but thatco-administration of a second polynucleotides, primary construct, ormmRNA encoding CYP7A1 would continue to drive the cholesterol outthrough bile thereby avoiding the fatty liver symptoms currently seenwith known therapeutics.

In addition to delivering at least LDLR or a PCSK9 LDLR mutant alongwith CYP7A1 polynucleotides, primary constructs or mmRNAs, it is furtherexpected that delivering an additional drug such as a statin would bevery synergistic to the LDLR-CYP7A1 therapy described herein.Consequently, cholesterol excretion would be promoted, new formationwould be prevented and transport from the plasma would be increased.

In one embodiment, a mix of mmRNA would be titrated along thecholesterol homeostasis pathway to promote mobilization of cholesterolout of the body.

In another embodiment, bile acid sequestrants or fat soluble vitaminsmay be co-administered.

It is further appreciated that certain features of the presentdisclosure, which are, for clarity, described in the context of separateembodiments, can also be provided in combination in a single embodiment.Conversely, various features of the present disclosure which are, forbrevity, described in the context of a single embodiment, can also beprovided separately or in any suitable subcombination.

The details of various embodiments of the invention are set forth in thedescription below. Other features, objects, and advantages of theinvention will be apparent from the description and the drawings, andfrom the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objects, features and advantages will beapparent from the following description of particular embodiments of theinvention, as illustrated in the accompanying drawings in which likereference characters refer to the same parts throughout the differentviews. The drawings are not necessarily to scale, emphasis instead beingplaced upon illustrating the principles of various embodiments of theinvention.

FIG. 1 is a schematic of a primary construct of the present invention.

FIG. 2 shows the pathway of cholesterol trafficking in a liver cell.

FIG. 3 is a flow cytometry plot of low density lipoprotein receptor(LDLR) modified mRNA.

FIG. 4 is a flow cytometry plot of LDLR modified mRNA.

FIGS. 5A-5E are graphs of LDLR Expression. FIG. 5A shows LDL ReceptorExpression of cells compared to LDLR mRNA added. FIG. 5B shows LDLReceptor Expression of cells post transfection. FIG. 5C shows thesaturation of BODIPY® labeled LRL. FIG. 5D shows the binding affinity ofBODIPY-LDL to cells. FIG. 5E shows the total cholesterol content of eachfraction.

FIG. 6 is a bioanalyzer image of LDLR modified mRNA product. Lane 1,size markers in nucleotides; Lane 2, LDLR modified mRNA.

FIG. 7 is a flow cytometry plot of 800 ng LDLR modified mRNA transfectedin HEK293 cells.

FIG. 8 is a flow cytometry plot of LDLR modified mRNA transfected inHEK293 cells.

FIGS. 9A and 9B show the cholesterol level in serum. FIG. 9A shows theabsorbance profile of FPLC fractions from pooled LDLR knock out mice(Upper panel) and wild type mice (lower panel). FIG. 9B shows the totalcholesterol content of each fraction.

FIG. 10 is a flow cytometry plot of variant LDLR modified mRNAtransfected in HEK293 cells.

FIG. 11 is a flow cytometry plot of variant LDLR modified mRNAtransfected in HEK293 cells with or without PCSK9.

FIGS. 12A and 12B are flow cytometry plots of transfected variant LDLRmodified mRNA. FIG. 12A shows contour plots of the binding of BODIPY-LDLto LDLR mRNA transfected cells. FIG. 12B shows the half-maximal cellassociation of BODIPY-LDL.

FIGS. 13A-13G show the effect on half-life after transfection with LDLRmRNA.

FIG. 13A shows wild-type LDLR mRNA. FIG. 13B shows a LDLR mRNA encodinga variant LDLR with 4 amino acid substitutions (N316A, E317A, D331A andY336A). FIG. 13C shows a LDLR mRNA encoding a variant LDLR with 1 aminoacid substitution, Y336A.

FIG. 13D shows a LDLR mRNA encoding a variant LDLR with 1 amino acidsubstitution, E317A. FIG. 13E shows a LDLR mRNA encoding a variant LDLRwith 1 amino acid substitution, N316A. FIG. 13F shows a LDLR mRNAencoding a variant LDLR with 1 amino acid substitution, L339D. FIG. 13Gshows a LDLR mRNA encoding a variant LDLR with 1 amino acidsubstitution, D331E.

FIG. 14 shows the effect on cell surface LDLR when the amount of PCSK isvaried.

DETAILED DESCRIPTION

It is of great interest in the fields of therapeutics, diagnostics,reagents and for biological assays to be able to deliver a nucleic acid,e.g., a ribonucleic acid (RNA) inside a cell, whether in vitro, in vivo,in situ or ex vivo, such as to cause intracellular translation of thenucleic acid and production of an encoded polypeptide of interest. Ofparticular importance is the delivery and function of a non-integrativepolynucleotide.

Described herein are compositions (including pharmaceuticalcompositions) and methods for the design, preparation, manufactureand/or formulation of polynucleotides encoding one or more polypeptidesof interest. Also provided are systems, processes, devices and kits forthe selection, design and/or utilization of the polynucleotides encodingthe polypeptides of interest described herein.

According to the present invention, these polynucleotides are preferablymodified as to avoid the deficiencies of other polypeptide-encodingmolecules of the art. Hence these polynucleotides are referred to asmodified mRNA or mmRNA.

Provided herein, in part, are polynucleotides, primary constructs and/ormmRNA encoding polypeptides of interest which have been designed toimprove one or more of the stability and/or clearance in tissues,receptor uptake and/or kinetics, cellular access by the compositions,engagement with translational machinery, mRNA half-life, translationefficiency, immune evasion, protein production capacity, secretionefficiency (when applicable), accessibility to circulation, proteinhalf-life and/or modulation of a cell's status, function and/oractivity. Specifically, the polynucleotides, primary constructs and/ormmRNA of the present invention are useful in altering cholesterol levelsor cholesterol trafficking in an organism, particularly human patients.

According to the present invention, the pathways associated withcholesterol trafficking are modulated by providing one or morepolypeptides (including enzymes) which alter either the concentrationsof cholesterol, its processing or transport.

In one embodiment, the transport of LDL cholesterol from plasma to livercells is increased by providing the cell with either more receptormolecules or by minimizing the destruction of the LDL receptor. In thefirst instance a polynucleotide, primary construct or mmRNA is providedwhich encodes LDL receptor. In the second instance, a mutant form of LDLreceptor is encoded by the polynucleotide, primary construct or mmRNA.Such mutant LDL receptors (LDL-R or LDLR) would be deficient in some wayin their binding of PCSK-9. The binding site of PCSK-9 has beenpreviously localized to the EGF-A (or EGF-like repeat) domain of LDLR(see e.g., Kwon et al. Molecular Basis for LDL receptor recognition byPCSK9. PNAS. 2008 105(6), 1820-1825; the contents of which is hereinincorporated by reference in its entirety). Accordingly, a PCSK9 bindingdeficient LDLR would bring cholesterol into the hepatocyte.

In one embodiment, a polynucleotide, primary construct or mmRNA mayencode a mutant LDLR which is deficient in binding to PCSK-9.

In one embodiment, a polynucleotide, primary construct or mmRNA mayencode a mutant LDLR which comprises at least one amino acid mutation inthe PCSK-9 binding site. The mutant LDLR may comprise one, two, three,four, five, six, seven, eight, nine, ten or more than ten mutations.

In one embodiment, the mutant LDLR may comprise at least one amino acidmutation in the EGF-A domain (also known as the EGF-like repeat domain)of LDLR. As a non-limiting example, the EGF-A domain is located in aregion of LDLR comprising amino acids 314-393. As another non-limitingexample, the EGF-A domain is a region of SEQ ID NO: 19 comprising aminoacids 314-393.

In one embodiment, a polynucleotide, primary construct or mmRNA mayencode a mutant LDLR which comprises at least one amino acid mutation inthe EGF-like 1 domain. The EGF-like 1 domain may be located in a regionof LDLR comprising amino acids 314-353. As a non-limiting example, theEGF-like 1 domain is a region of SEQ ID NO: 19 comprising amino acids314-353.

In one embodiment, a polynucleotide, primary construct or mmRNA mayencode a mutant LDLR which comprises at least one amino acid mutation inthe EGF-like 2 domain. The EGF-like 2 domain may be located in a regionof LDLR comprising amino acids 353-393. As a non-limiting example, theEGF-like 2 domain is a region of SEQ ID NO: 19 comprising amino acids353-393.

In one embodiment, a polynucleotide, primary construct or mmRNA mayencode a PCSK-9 binding deficient mutant LDLR. The PCSK-9 bindingdeficient mutant LDLR may comprise at least one amino acid mutation inthe region of SEQ ID NO: 19 comprising amino acids 314-393. As anon-limiting example, at least one mutation may be located between aminoacids 314-353. As another non-limiting example, at least one mutationmay be located between amino acids 315-340. As yet another non-limitingexample, at least one mutation may be located between amino acids354-393.

In one embodiment, a polynucleotide, primary construct or mmRNA mayencode a PCSK-9 binding deficient mutant LDLR comprising at least onemutation in the PCSK-9 binding region. The mutation may be located inthe region of SEQ ID NO: 19 comprising amino acids 314-393. Asnon-limiting examples of regions of mutations, the mutation may belocated in the region of 314-353, 315-340 and 354-393. As anothernon-limiting example, the mutation may be at position 316, 317, 331, 336or 339. As yet another non-limiting example, the mutant LDLR maycomprise a mutation at position 316, 317, 331 and 336.

In one embodiment, a polynucleotide, primary construct or mmRNA mayencode a PCSK-9 binding deficient mutant LDLR comprising at least onemutation at an amino acid position such as, but not limited to, 316,317, 331, 336 and/or 339. As a non-limiting example, the PCSK-9 bindingdeficient mutant LDLR may comprise at least one of the mutations N316A,E317A, D331A, D331E, Y336A and/or L339D where “N316A” means Asparagineat position 316 is replaced with Alanine. As another non-limitingexample, the PCSK-9 binding deficient mutant LDLR may comprise themutations N316A, E317A, D331A and Y336A.

In one embodiment, a polynucleotide, primary construct or mmRNA mayencode a PCSK-9 binding deficient mutant LDLR comprising four mutationsat amino acid positions such as, but not limited to, 316, 317, 331, 336and/or 339. As a non-limiting example, the PCSK-9 binding deficientmutant LDLR may comprise any four of the mutations such as N316A, E317A,D331A, D331E, Y336A and/or L339D where “N316A” means Asparagine atposition 316 is replaced with Alanine. As another non-limiting example,the PCSK-9 binding deficient mutant LDLR may comprise the mutationsN316A, E317A, D331A and Y336A.

In one embodiment, the hepatocyte is provided with one or morepolynucleotides, primary constructs, or mmRNA encoding and/or whichoverexpresses CYP7A1. CYP7A1 is the rate limiting enzyme for bile acidsynthesis, and promotes removal of the incoming cholesterol. There arehumans with CYP7A1 mutations that are associated with high plasmalow-density lipoprotein (LDL) and hepatic cholesterol content, as wellas deficient bile acid excretion.

In one embodiment, two polynucleotides, primary constructs, or mmRNA aredelivered resulting in lower plasma cholesterol and concomitant enhancedcholesterol disposal.

In one embodiment, the one or more polynucleotides, primary constructsor mmRNA are modified in the 3′UTR to contain a microRNA binding site orseed. In this embodiment, the CYP7A1 polynucleotide, having a normalhalf life of approximately 30 minutes, may be madetranscription-dependent by miR-destabilization (specifically theubiquitous miR122a in hepatocytes). According to this embodiment, amiR122a binding site may be incorporated into the 3′UTR of the mmRNArendering the transcript less stable. This would allow, depending on thenumber of binding sites engineered into the construct, the titration ofstability and therefore allow for control of expression of the encodedCYP7A1 enzyme. The polynucleotides, primary constructs primaryconstructs or mmRNA encoding CYP7A1 may also be useful in creating mousemodels useful in proof of concept studies and basic research. Thesestudies would be analogous to producing dose dependent gene therapy.

In one embodiment, treatment regimes may be designed for rare diseaseswhere patients present with CYP7A1 polymorphisms that are hyporesponsiveto statins. In this instance, studies of effective compositions of thepresent invention may be completed quickly and based on diet-basedchallenges. As such the compositions of the present invention are usefulin the study and treatment of disease involving cholesterol relateddiseases, both rare and prevalent.

In one embodiment, the compositions of the present invention may beadministered along with other drug compounds. Such other drugs includespecifically statins. Examples of statins include, but are not limitedto, atorvastatin, cerivastatin, fluvastatin, lovastatin, mevastatin,pitavastatin, pravastatin, simvastatin, and combinations thereof.

According to the present invention, the compositions comprisingpolynucleotides, primary constructs or mmRNA are useful in treatingdiseases such as rare non-alcoholic fatty liver disease. In treatingthis disorder, it is contemplated that any therapeutic that driveshepatic cholesterol to its natural sink (out of the body through biles)would have superior treatment outcomes. Consequently, it is contemplatedthat administration of polynucleotides, primary constructs or mmRNAencoding LDLR would increase cholesterol in the hepatocyte but thatco-administration of a second polynucleotides, primary construct, ormmRNA encoding CYP7A1 would continue to drive the cholesterol outthrough bile thereby avoiding the fatty liver symptoms currently seenwith known therapeutics.

In addition to delivering at least LDLR or a PCSK9 LDLR mutant alongwith CYP7A1 polynucleotides, primary constructs or mmRNAs, it is furtherexpected that delivering an additional drug such as a statin would bevery synergistic to the LDLR-CYP7A1 therapy described herein.Consequently, cholesterol excretion would be promoted, new formationwould be prevented and transport from the plasma would be increased.

In one embodiment, a mix of mmRNA would be titrated along thecholesterol homeostasis pathway to promote mobilization of cholesterolout of the body.

In another embodiment, bile acid sequestrants or fat soluble vitaminsmay be co-administered.

It is further appreciated that certain features of the presentdisclosure, which are, for clarity, described in the context of separateembodiments, can also be provided in combination in a single embodiment.Conversely, various features of the present disclosure which are, forbrevity, described in the context of a single embodiment, can also beprovided separately or in any suitable subcombination.

I. Compositions of the Invention (mmRNA)

The present invention provides nucleic acid molecules, specificallypolynucleotides, primary constructs and/or mmRNA which encode one ormore polypeptides of interest. The term “nucleic acid,” in its broadestsense, includes any compound and/or substance that comprise a polymer ofnucleotides. These polymers are often referred to as polynucleotides.Exemplary nucleic acids or polynucleotides of the invention include, butare not limited to, ribonucleic acids (RNAs), deoxyribonucleic acids(DNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs),peptide nucleic acids (PNAs), locked nucleic acids (LNAs, including LNAhaving a (3-D-ribo configuration, α-LNA having an α-L-ribo configuration(a diastereomer of LNA), 2′-amino-LNA having a 2′-aminofunctionalization, and 2′-amino-α-LNA having a 2′-aminofunctionalization) or hybrids thereof.

In preferred embodiments, the nucleic acid molecule is a messenger RNA(mRNA). As used herein, the term “messenger RNA” (mRNA) refers to anypolynucleotide, such as a synthetic polynucleotide, which encodes apolypeptide of interest and which is capable of being translated toproduce the encoded polypeptide of interest in vitro, in vivo, in situor ex vivo.

Traditionally, the basic components of an mRNA molecule include at leasta coding region, a 5′UTR, a 3′UTR, a 5′ cap and a poly-A tail. Buildingon this wild type modular structure, the present invention expands thescope of functionality of traditional mRNA molecules by providingpolynucleotides or primary RNA constructs which maintain a modularorganization, but which comprise one or more structural and/or chemicalmodifications or alterations which impart useful properties to thereprogramming polynucleotides including, in some embodiments, the lackof a substantial induction of the innate immune response of a cell intowhich the polynucleotide is introduced. As such, modified mRNA moleculesof the present invention, such as synthetic modified mRNA molecules, aretermed “mmRNA.” As used herein, a “structural” feature or modificationis one in which two or more linked nucleotides are inserted, deleted,duplicated, inverted or randomized in a polynucleotide, primaryconstruct or mmRNA without significant chemical modification to thenucleotides themselves. Because chemical bonds will necessarily bebroken and reformed to effect a structural modification, structuralmodifications are of a chemical nature and hence are chemicalmodifications. However, structural modifications will result in adifferent sequence of nucleotides. For example, the polynucleotide“ATCG” may be chemically modified to “AT-5meC-G”. The samepolynucleotide may be structurally modified from “ATCG” to “ATCCCG”.Here, the dinucleotide “CC” has been inserted, resulting in a structuralmodification to the polynucleotide.

mmRNA Architecture

The mmRNA of the present invention are distinguished from wild type mRNAin their functional and/or structural design features which serve to, asevidenced herein, overcome existing problems of effective polypeptideproduction using nucleic acid-based therapeutics.

FIG. 1 shows a representative polynucleotide primary construct 100 ofthe present invention. As used herein, the term “primary construct” or“primary mRNA construct” refers to a polynucleotide transcript whichencodes one or more polypeptides of interest and which retainssufficient structural and/or chemical features to allow the polypeptideof interest encoded therein to be translated. Primary constructs may bepolynucleotides of the invention. When structurally or chemicallymodified, the primary construct may be referred to as an mmRNA.

Returning to FIG. 1, the primary construct 100 here contains a firstregion of linked nucleotides 102 that is flanked by a first flankingregion 104 and a second flaking region 106. As used herein, the “firstregion” may be referred to as a “coding region” or “region encoding” orsimply the “first region.” This first region may include, but is notlimited to, the encoded polypeptide of interest. The polypeptide ofinterest may comprise at its 5′ terminus one or more signal sequencesencoded by a signal sequence region 103. The flanking region 104 maycomprise a region of linked nucleotides comprising one or more completeor incomplete 5′ UTRs sequences. The flanking region 104 may alsocomprise a 5′ terminal cap 108. The second flanking region 106 maycomprise a region of linked nucleotides comprising one or more completeor incomplete 3′ UTRs. The flanking region 106 may also comprise a 3′tailing sequence 110.

Bridging the 5′ terminus of the first region 102 and the first flankingregion 104 is a first operational region 105. Traditionally thisoperational region comprises a Start codon. The operational region mayalternatively comprise any translation initiation sequence or signalincluding a Start codon.

Bridging the 3′ terminus of the first region 102 and the second flankingregion 106 is a second operational region 107. Traditionally thisoperational region comprises a Stop codon. The operational region mayalternatively comprise any translation initiation sequence or signalincluding a Stop codon. According to the present invention, multipleserial stop codons may also be used.

Generally, the shortest length of the first region of the primaryconstruct of the present invention can be the length of a nucleic acidsequence that is sufficient to encode for a dipeptide, a tripeptide, atetrapeptide, a pentapeptide, a hexapeptide, a heptapeptide, anoctapeptide, a nonapeptide, or a decapeptide. In another embodiment, thelength may be sufficient to encode a peptide of 2-30 amino acids, e.g.5-30, 10-30, 2-25, 5-25, 10-25, or 10-20 amino acids. The length may besufficient to encode for a peptide of at least 11, 12, 13, 14, 15, 17,20, 25 or 30 amino acids, or a peptide that is no longer than 40 aminoacids, e.g. no longer than 35, 30, 25, 20, 17, 15, 14, 13, 12, 11 or 10amino acids. Examples of dipeptides that the polynucleotide sequencescan encode or include, but are not limited to, carnosine and anserine.

Generally, the length of the first region encoding the polypeptide ofinterest of the present invention is greater than about 30 nucleotidesin length (e.g., at least or greater than about 35, 40, 45, 50, 55, 60,70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500,600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600,1,700, 1,800, 1,900, 2,000, 2,500, and 3,000, 4,000, 5,000, 6,000,7,000, 8,000, 9,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000,70,000, 80,000, 90,000 or up to and including 100,000 nucleotides). Asused herein, the “first region” may be referred to as a “coding region”or “region encoding” or simply the “first region.”

In some embodiments, the polynucleotide, primary construct, or mmRNAincludes from about 30 to about 100,000 nucleotides (e.g., from 30 to50, from 30 to 100, from 30 to 250, from 30 to 500, from 30 to 1,000,from 30 to 1,500, from 30 to 3,000, from 30 to 5,000, from 30 to 7,000,from 30 to 10,000, from 30 to 25,000, from 30 to 50,000, from 30 to70,000, from 100 to 250, from 100 to 500, from 100 to 1,000, from 100 to1,500, from 100 to 3,000, from 100 to 5,000, from 100 to 7,000, from 100to 10,000, from 100 to 25,000, from 100 to 50,000, from 100 to 70,000,from 100 to 100,000, from 500 to 1,000, from 500 to 1,500, from 500 to2,000, from 500 to 3,000, from 500 to 5,000, from 500 to 7,000, from 500to 10,000, from 500 to 25,000, from 500 to 50,000, from 500 to 70,000,from 500 to 100,000, from 1,000 to 1,500, from 1,000 to 2,000, from1,000 to 3,000, from 1,000 to 5,000, from 1,000 to 7,000, from 1,000 to10,000, from 1,000 to 25,000, from 1,000 to 50,000, from 1,000 to70,000, from 1,000 to 100,000, from 1,500 to 3,000, from 1,500 to 5,000,from 1,500 to 7,000, from 1,500 to 10,000, from 1,500 to 25,000, from1,500 to 50,000, from 1,500 to 70,000, from 1,500 to 100,000, from 2,000to 3,000, from 2,000 to 5,000, from 2,000 to 7,000, from 2,000 to10,000, from 2,000 to 25,000, from 2,000 to 50,000, from 2,000 to70,000, and from 2,000 to 100,000).

According to the present invention, the first and second flankingregions may range independently from 15-1,000 nucleotides in length(e.g., greater than 30, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140,160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, and 900nucleotides or at least 30, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120,140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900,and 1,000 nucleotides).

According to the present invention, the tailing sequence may range fromabsent to 500 nucleotides in length (e.g., at least 60, 70, 80, 90, 120,140, 160, 180, 200, 250, 300, 350, 400, 450, or 500 nucleotides). Wherethe tailing region is a polyA tail, the length may be determined inunits of or as a function of polyA Binding Protein binding. In thisembodiment, the polyA tail is long enough to bind at least 4 monomers ofPolyA Binding Protein. PolyA Binding Protein monomers bind to stretchesof approximately 38 nucleotides. As such, it has been observed thatpolyA tails of about 80 nucleotides and 160 nucleotides are functional.

According to the present invention, the capping region may comprise asingle cap or a series of nucleotides forming the cap. In thisembodiment the capping region may be from 1 to 10, e.g. 2-9, 3-8, 4-7,1-5, 5-10, or at least 2, or 10 or fewer nucleotides in length. In someembodiments, the cap is absent.

According to the present invention, the first and second operationalregions may range from 3 to 40, e.g., 5-30, 10-20, 15, or at least 4, or30 or fewer nucleotides in length and may comprise, in addition to aStart and/or Stop codon, one or more signal and/or restrictionsequences.

Cyclic mmRNA

According to the present invention, a primary construct or mmRNA may becyclized, or concatemerized, to generate a translation competentmolecule to assist interactions between poly-A binding proteins and5′-end binding proteins. The mechanism of cyclization orconcatemerization may occur through at least 3 different routes: 1)chemical, 2) enzymatic, and 3) ribozyme catalyzed. The newly formed5′-/3′-linkage may be intramolecular or intermolecular.

In the first route, the 5′-end and the 3′-end of the nucleic acidcontain chemically reactive groups that, when close together, form a newcovalent linkage between the 5′-end and the 3′-end of the molecule. The5′-end may contain an NETS-ester reactive group and the 3′-end maycontain a 3′-amino-terminated nucleotide such that in an organic solventthe 3′-amino-terminated nucleotide on the 3′-end of a synthetic mRNAmolecule will undergo a nucleophilic attack on the 5′-NHS-ester moietyforming a new 5′-/3′-amide bond.

In the second route, T4 RNA ligase may be used to enzymatically link a5′-phosphorylated nucleic acid molecule to the 3′-hydroxyl group of anucleic acid forming a new phosphorodiester linkage. In an examplereaction, 1 μg of a nucleic acid molecule is incubated at 37° C. for 1hour with 1-10 units of T4 RNA ligase (New England Biolabs, Ipswich,Mass.) according to the manufacturer's protocol. The ligation reactionmay occur in the presence of a split oligonucleotide capable ofbase-pairing with both the 5′- and 3′-region in juxtaposition to assistthe enzymatic ligation reaction.

In the third route, either the 5′- or 3′-end of the cDNA templateencodes a ligase ribozyme sequence such that during in vitrotranscription, the resultant nucleic acid molecule can contain an activeribozyme sequence capable of ligating the 5′-end of a nucleic acidmolecule to the 3′-end of a nucleic acid molecule. The ligase ribozymemay be derived from the Group I Intron, Group I Intron, Hepatitis DeltaVirus, Hairpin ribozyme or may be selected by SELEX (systematicevolution of ligands by exponential enrichment). The ribozyme ligasereaction may take 1 to 24 hours at temperatures between 0 and 37° C.

mmRNA Multimers

According to the present invention, multiple distinct polynucleotides,primary constructs or mmRNA may be linked together through the 3′-endusing nucleotides which are modified at the 3′-terminus. Chemicalconjugation may be used to control the stoichiometry of delivery intocells. For example, the glyoxylate cycle enzymes, isocitrate lyase andmalate synthase, may be supplied into HepG2 cells at a 1:1 ratio toalter cellular fatty acid metabolism. This ratio may be controlled bychemically linking polynucleotides, primary constructs or mmRNA using a3′-azido terminated nucleotide on one polynucleotide, primary constructor mmRNA species and a C5-ethynyl or alkynyl-containing nucleotide onthe opposite polynucleotide, primary construct or mmRNA species. Themodified nucleotide is added post-transcriptionally using terminaltransferase (New England Biolabs, Ipswich, Mass.) according to themanufacturer's protocol. After the addition of the 3′-modifiednucleotide, the two polynucleotide, primary construct or mmRNA speciesmay be combined in an aqueous solution, in the presence or absence ofcopper, to form a new covalent linkage via a click chemistry mechanismas described in the literature.

In another example, more than two polynucleotides may be linked togetherusing a functionalized linker molecule. For example, a functionalizedsaccharide molecule may be chemically modified to contain multiplechemical reactive groups (SH—, NH₂—, N₃, etc. . . . ) to react with thecognate moiety on a 3′-functionalized mRNA molecule (i.e., a3′-maleimide ester, 3′-NHS-ester, alkynyl). The number of reactivegroups on the modified saccharide can be controlled in a stoichiometricfashion to directly control the stoichiometric ratio of conjugatedpolynucleotide, primary construct or mmRNA.

mmRNA Conjugates and Combinations

In order to further enhance protein production, primary constructs ormmRNA of the present invention can be designed to be conjugated to otherpolynucleotides, dyes, intercalating agents (e.g. acridines),cross-linkers (e.g. psoralene, mitomycin C), porphyrins (TPPC4,texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g.,phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA),alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K),MPEG, [MPEG]₂, polyamino, alkyl, substituted alkyl, radiolabeledmarkers, enzymes, haptens (e.g. biotin), transport/absorptionfacilitators (e.g., aspirin, vitamin E, folic acid), syntheticribonucleases, proteins, e.g., glycoproteins, or peptides, e.g.,molecules having a specific affinity for a co-ligand, or antibodiese.g., an antibody, that binds to a specified cell type such as a cancercell, endothelial cell, or bone cell, hormones and hormone receptors,non-peptidic species, such as lipids, lectins, carbohydrates, vitamins,cofactors, or a drug.

Conjugation may result in increased stability and/or half life and maybe particularly useful in targeting the polynucleotides, primaryconstructs or mmRNA to specific sites in the cell, tissue or organism.

According to the present invention, the mmRNA or primary constructs maybe administered with, or further encode one or more of RNAi agents,siRNAs, shRNAs, miRNAs, miRNA binding sites, antisense RNAs, ribozymes,catalytic DNA, tRNA, RNAs that induce triple helix formation, aptamersor vectors, and the like.

Bifunctional mmRNA

In one embodiment of the invention are bifunctional polynucleotides(e.g., bifunctional primary constructs or bifunctional mmRNA). As thename implies, bifunctional polynucleotides are those having or capableof at least two functions. These molecules may also by convention bereferred to as multi-functional.

The multiple functionalities of bifunctional polynucleotides may beencoded by the RNA (the function may not manifest until the encodedproduct is translated) or may be a property of the polynucleotideitself. It may be structural or chemical. Bifunctional modifiedpolynucleotides may comprise a function that is covalently orelectrostatically associated with the polynucleotides. Further, the twofunctions may be provided in the context of a complex of a mmRNA andanother molecule.

Bifunctional polynucleotides may encode peptides which areanti-proliferative. These peptides may be linear, cyclic, constrained orrandom coil. They may function as aptamers, signaling molecules, ligandsor mimics or mimetics thereof. Anti-proliferative peptides may, astranslated, be from 3 to 50 amino acids in length. They may be 5-40,10-30, or approximately 15 amino acids long. They may be single chain,multichain or branched and may form complexes, aggregates or anymulti-unit structure once translated.

Noncoding Polynucleotides and Primary Constructs

As described herein, provided are polynucleotides and primary constructshaving sequences that are partially or substantially not translatable,e.g., having a noncoding region. Such noncoding region may be the “firstregion” of the primary construct. Alternatively, the noncoding regionmay be a region other than the first region. Such molecules aregenerally not translated, but can exert an effect on protein productionby one or more of binding to and sequestering one or more translationalmachinery components such as a ribosomal protein or a transfer RNA(tRNA), thereby effectively reducing protein expression in the cell ormodulating one or more pathways or cascades in a cell which in turnalters protein levels. The polynucleotide or primary construct maycontain or encode one or more long noncoding RNA (lncRNA, or lincRNA) orportion thereof, a small nucleolar RNA (sno-RNA), micro RNA (miRNA),small interfering RNA (siRNA) or Piwi-interacting RNA (piRNA).

Polypeptides of Interest

According to the present invention, the primary construct is designed toencode one or more polypeptides of interest or fragments thereof. Apolypeptide of interest may include, but is not limited to, wholepolypeptides, a plurality of polypeptides or fragments of polypeptides,which independently may be encoded by one or more nucleic acids, aplurality of nucleic acids, fragments of nucleic acids or variants ofany of the aforementioned. As used herein, the term “polypeptides ofinterest” refers to any polypeptides which are selected to be encoded inthe primary construct of the present invention. As used herein,“polypeptide” means a polymer of amino acid residues (natural orunnatural) linked together most often by peptide bonds. The term, asused herein, refers to proteins, polypeptides, and peptides of any size,structure, or function. In some instances the polypeptide encoded issmaller than about 50 amino acids and the polypeptide is then termed apeptide. If the polypeptide is a peptide, it will be at least about 2,3, 4, or at least 5 amino acid residues long. Thus, polypeptides includegene products, naturally occurring polypeptides, synthetic polypeptides,homologs, orthologs, paralogs, fragments and other equivalents,variants, and analogs of the foregoing. A polypeptide may be a singlemolecule or may be a multi-molecular complex such as a dimer, trimer ortetramer. They may also comprise single chain or multichain polypeptidessuch as antibodies or insulin and may be associated or linked. Mostcommonly disulfide linkages are found in multichain polypeptides. Theterm polypeptide may also apply to amino acid polymers in which one ormore amino acid residues are an artificial chemical analogue of acorresponding naturally occurring amino acid.

The term “polypeptide variant” refers to molecules which differ in theiramino acid sequence from a native or reference sequence. The amino acidsequence variants may possess substitutions, deletions, and/orinsertions at certain positions within the amino acid sequence, ascompared to a native or reference sequence. Ordinarily, variants willpossess at least about 50% identity (homology) to a native or referencesequence, and preferably, they will be at least about 80%, morepreferably at least about 90% identical (homologous) to a native orreference sequence.

In some embodiments “variant mimics” are provided. As used herein, theterm “variant mimic” is one which contains one or more amino acids whichwould mimic an activated sequence. For example, glutamate may serve as amimic for phosphoro-threonine and/or phosphoro-serine. Alternatively,variant mimics may result in deactivation or in an inactivated productcontaining the mimic, e.g., phenylalanine may act as an inactivatingsubstitution for tyrosine; or alanine may act as an inactivatingsubstitution for serine.

“Homology” as it applies to amino acid sequences is defined as thepercentage of residues in the candidate amino acid sequence that areidentical with the residues in the amino acid sequence of a secondsequence after aligning the sequences and introducing gaps, ifnecessary, to achieve the maximum percent homology. Methods and computerprograms for the alignment are well known in the art. It is understoodthat homology depends on a calculation of percent identity but maydiffer in value due to gaps and penalties introduced in the calculation.

By “homologs” as it applies to polypeptide sequences means thecorresponding sequence of other species having substantial identity to asecond sequence of a second species.

“Analogs” is meant to include polypeptide variants which differ by oneor more amino acid alterations, e.g., substitutions, additions ordeletions of amino acid residues that still maintain one or more of theproperties of the parent or starting polypeptide.

The present invention contemplates several types of compositions whichare polypeptide based including variants and derivatives. These includesubstitutional, insertional, deletion and covalent variants andderivatives. The term “derivative” is used synonymously with the term“variant” but generally refers to a molecule that has been modifiedand/or changed in any way relative to a reference molecule or startingmolecule.

As such, mmRNA encoding polypeptides containing substitutions,insertions and/or additions, deletions and covalent modifications withrespect to reference sequences, in particular the polypeptide sequencesdisclosed herein, are included within the scope of this invention. Forexample, sequence tags or amino acids, such as one or more lysines, canbe added to the peptide sequences of the invention (e.g., at theN-terminal or C-terminal ends). Sequence tags can be used for peptidepurification or localization. Lysines can be used to increase peptidesolubility or to allow for biotinylation. Alternatively, amino acidresidues located at the carboxy and amino terminal regions of the aminoacid sequence of a peptide or protein may optionally be deletedproviding for truncated sequences. Certain amino acids (e.g., C-terminalor N-terminal residues) may alternatively be deleted depending on theuse of the sequence, as for example, expression of the sequence as partof a larger sequence which is soluble, or linked to a solid support.

“Substitutional variants” when referring to polypeptides are those thathave at least one amino acid residue in a native or starting sequenceremoved and a different amino acid inserted in its place at the sameposition. The substitutions may be single, where only one amino acid inthe molecule has been substituted, or they may be multiple, where two ormore amino acids have been substituted in the same molecule.

As used herein the term “conservative amino acid substitution” refers tothe substitution of an amino acid that is normally present in thesequence with a different amino acid of similar size, charge, orpolarity. Examples of conservative substitutions include thesubstitution of a non-polar (hydrophobic) residue such as isoleucine,valine and leucine for another non-polar residue. Likewise, examples ofconservative substitutions include the substitution of one polar(hydrophilic) residue for another such as between arginine and lysine,between glutamine and asparagine, and between glycine and serine.Additionally, the substitution of a basic residue such as lysine,arginine or histidine for another, or the substitution of one acidicresidue such as aspartic acid or glutamic acid for another acidicresidue are additional examples of conservative substitutions. Examplesof non-conservative substitutions include the substitution of anon-polar (hydrophobic) amino acid residue such as isoleucine, valine,leucine, alanine, methionine for a polar (hydrophilic) residue such ascysteine, glutamine, glutamic acid or lysine and/or a polar residue fora non-polar residue.

“Insertional variants” when referring to polypeptides are those with oneor more amino acids inserted immediately adjacent to an amino acid at aparticular position in a native or starting sequence. “Immediatelyadjacent” to an amino acid means connected to either the alpha-carboxyor alpha-amino functional group of the amino acid.

“Deletional variants” when referring to polypeptides are those with oneor more amino acids in the native or starting amino acid sequenceremoved. Ordinarily, deletional variants will have one or more aminoacids deleted in a particular region of the molecule.

“Covalent derivatives” when referring to polypeptides includemodifications of a native or starting protein with an organicproteinaceous or non-proteinaceous derivatizing agent, and/orpost-translational modifications. Covalent modifications aretraditionally introduced by reacting targeted amino acid residues of theprotein with an organic derivatizing agent that is capable of reactingwith selected side-chains or terminal residues, or by harnessingmechanisms of post-translational modifications that function in selectedrecombinant host cells. The resultant covalent derivatives are useful inprograms directed at identifying residues important for biologicalactivity, for immunoassays, or for the preparation of anti-proteinantibodies for immunoaffinity purification of the recombinantglycoprotein. Such modifications are within the ordinary skill in theart and are performed without undue experimentation.

Certain post-translational modifications are the result of the action ofrecombinant host cells on the expressed polypeptide. Glutaminyl andasparaginyl residues are frequently post-translationally deamidated tothe corresponding glutamyl and aspartyl residues. Alternatively, theseresidues are deamidated under mildly acidic conditions. Either form ofthese residues may be present in the polypeptides produced in accordancewith the present invention.

Other post-translational modifications include hydroxylation of prolineand lysine, phosphorylation of hydroxyl groups of seryl or threonylresidues, methylation of the alpha-amino groups of lysine, arginine, andhistidine side chains (T. E. Creighton, Proteins: Structure andMolecular Properties, W.H. Freeman & Co., San Francisco, pp. 79-86(1983)).

“Features” when referring to polypeptides are defined as distinct aminoacid sequence-based components of a molecule. Features of thepolypeptides encoded by the mmRNA of the present invention includesurface manifestations, local conformational shape, folds, loops,half-loops, domains, half-domains, sites, termini or any combinationthereof.

As used herein when referring to polypeptides the term “surfacemanifestation” refers to a polypeptide based component of a proteinappearing on an outermost surface.

As used herein when referring to polypeptides the term “localconformational shape” means a polypeptide based structural manifestationof a protein which is located within a definable space of the protein.

As used herein when referring to polypeptides the term “fold” refers tothe resultant conformation of an amino acid sequence upon energyminimization. A fold may occur at the secondary or tertiary level of thefolding process. Examples of secondary level folds include beta sheetsand alpha helices. Examples of tertiary folds include domains andregions formed due to aggregation or separation of energetic forces.Regions formed in this way include hydrophobic and hydrophilic pockets,and the like.

As used herein the term “turn” as it relates to protein conformationmeans a bend which alters the direction of the backbone of a peptide orpolypeptide and may involve one, two, three or more amino acid residues.

As used herein when referring to polypeptides the term “loop” refers toa structural feature of a polypeptide which may serve to reverse thedirection of the backbone of a peptide or polypeptide. Where the loop isfound in a polypeptide and only alters the direction of the backbone, itmay comprise four or more amino acid residues. Oliva et al. haveidentified at least 5 classes of protein loops (J. Mol Biol 266 (4):814-830; 1997). Loops may be open or closed. Closed loops or “cyclic”loops may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acidsbetween the bridging moieties. Such bridging moieties may comprise acysteine-cysteine bridge (Cys-Cys) typical in polypeptides havingdisulfide bridges or alternatively bridging moieties may be non-proteinbased such as the dibromozylyl agents used herein.

As used herein when referring to polypeptides the term “half-loop”refers to a portion of an identified loop having at least half thenumber of amino acid resides as the loop from which it is derived. It isunderstood that loops may not always contain an even number of aminoacid residues. Therefore, in those cases where a loop contains or isidentified to comprise an odd number of amino acids, a half-loop of theodd-numbered loop will comprise the whole number portion or next wholenumber portion of the loop (number of amino acids of the loop/2+/−0.5amino acids). For example, a loop identified as a 7 amino acid loopcould produce half-loops of 3 amino acids or 4 amino acids(7/2=3.5+/−0.5 being 3 or 4).

As used herein when referring to polypeptides the term “domain” refersto a motif of a polypeptide having one or more identifiable structuralor functional characteristics or properties (e.g., binding capacity,serving as a site for protein-protein interactions).

As used herein when referring to polypeptides the term “half-domain”means a portion of an identified domain having at least half the numberof amino acid resides as the domain from which it is derived. It isunderstood that domains may not always contain an even number of aminoacid residues. Therefore, in those cases where a domain contains or isidentified to comprise an odd number of amino acids, a half-domain ofthe odd-numbered domain will comprise the whole number portion or nextwhole number portion of the domain (number of amino acids of thedomain/2+/−0.5 amino acids). For example, a domain identified as a 7amino acid domain could produce half-domains of 3 amino acids or 4 aminoacids (7/2=3.5+/−0.5 being 3 or 4). It is also understood thatsub-domains may be identified within domains or half-domains, thesesubdomains possessing less than all of the structural or functionalproperties identified in the domains or half domains from which theywere derived. It is also understood that the amino acids that compriseany of the domain types herein need not be contiguous along the backboneof the polypeptide (i.e., nonadjacent amino acids may fold structurallyto produce a domain, half-domain or subdomain).

As used herein when referring to polypeptides the terms “site” as itpertains to amino acid based embodiments is used synonymously with“amino acid residue” and “amino acid side chain.” A site represents aposition within a peptide or polypeptide that may be modified,manipulated, altered, derivatized or varied within the polypeptide basedmolecules of the present invention.

As used herein the terms “termini” or “terminus” when referring topolypeptides refers to an extremity of a peptide or polypeptide. Suchextremity is not limited only to the first or final site of the peptideor polypeptide but may include additional amino acids in the terminalregions. The polypeptide based molecules of the present invention may becharacterized as having both an N-terminus (terminated by an amino acidwith a free amino group (NH2)) and a C-terminus (terminated by an aminoacid with a free carboxyl group (COOH)). Proteins of the invention arein some cases made up of multiple polypeptide chains brought together bydisulfide bonds or by non-covalent forces (multimers, oligomers). Thesesorts of proteins will have multiple N- and C-termini. Alternatively,the termini of the polypeptides may be modified such that they begin orend, as the case may be, with a non-polypeptide based moiety such as anorganic conjugate.

Once any of the features have been identified or defined as a desiredcomponent of a polypeptide to be encoded by the primary construct ormmRNA of the invention, any of several manipulations and/ormodifications of these features may be performed by moving, swapping,inverting, deleting, randomizing or duplicating. Furthermore, it isunderstood that manipulation of features may result in the same outcomeas a modification to the molecules of the invention. For example, amanipulation which involved deleting a domain would result in thealteration of the length of a molecule just as modification of a nucleicacid to encode less than a full length molecule would.

Modifications and manipulations can be accomplished by methods known inthe art such as, but not limited to, site directed mutagenesis. Theresulting modified molecules may then be tested for activity using invitro or in vivo assays such as those described herein or any othersuitable screening assay known in the art.

According to the present invention, the polypeptides may comprise aconsensus sequence which is discovered through rounds ofexperimentation. As used herein a “consensus” sequence is a singlesequence which represents a collective population of sequences allowingfor variability at one or more sites.

As recognized by those skilled in the art, protein fragments, functionalprotein domains, and homologous proteins are also considered to bewithin the scope of polypeptides of interest of this invention. Forexample, provided herein is any protein fragment (meaning a polypeptidesequence at least one amino acid residue shorter than a referencepolypeptide sequence but otherwise identical) of a reference protein 10,20, 30, 40, 50, 60, 70, 80, 90, 100 or greater than 100 amino acids inlength. In another example, any protein that includes a stretch of about20, about 30, about 40, about 50, or about 100 amino acids which areabout 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about95%, or about 100% identical to any of the sequences described hereincan be utilized in accordance with the invention. In certainembodiments, a polypeptide to be utilized in accordance with theinvention includes 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations asshown in any of the sequences provided or referenced herein.

Encoded Polypeptides

The polynucleotides, primary constructs or mmRNA of the presentinvention may be designed to encode polypeptides of interest such aspeptides and proteins.

In one embodiment primary constructs or mmRNA may encode variantpolypeptides which have a certain identity with a reference polypeptidesequence. As used herein, a “reference polypeptide sequence” refers to astarting polypeptide sequence. Reference sequences may be wild typesequences or any sequence to which reference is made in the design ofanother sequence. A “reference polypeptide sequence” may be any encodingLDLR and/or CYP7a1 or variants thereof.

The term “identity” as known in the art, refers to a relationshipbetween the sequences of two or more peptides, as determined bycomparing the sequences. In the art, identity also means the degree ofsequence relatedness between peptides, as determined by the number ofmatches between strings of two or more amino acid residues. Identitymeasures the percent of identical matches between the smaller of two ormore sequences with gap alignments (if any) addressed by a particularmathematical model or computer program (i.e., “algorithms”). Identity ofrelated peptides can be readily calculated by known methods. Suchmethods include, but are not limited to, those described inComputational Molecular Biology, Lesk, A. M., ed., Oxford UniversityPress, New York, 1988; Biocomputing: Informatics and Genome Projects,Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis ofSequence Data, Part 1, Griffin, A. M., and Griffin, H. G., eds., HumanaPress, New Jersey, 1994; Sequence Analysis in Molecular Biology, vonHeinje, G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M.and Devereux, J., eds., M. Stockton Press, New York, 1991; and Carilloet al., SIAM J. Applied Math. 48, 1073 (1988).

In some embodiments, the polypeptide variant may have the same or asimilar activity as the reference polypeptide. Alternatively, thevariant may have an altered activity (e.g., increased or decreased)relative to a reference polypeptide. Generally, variants of a particularpolynucleotide or polypeptide of the invention will have at least about40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% but less than 100% sequence identity tothat particular reference polynucleotide or polypeptide as determined bysequence alignment programs and parameters described herein and known tothose skilled in the art. Such tools for alignment include those of theBLAST suite (Stephen F. Altschul, Thomas L. Madden, Alejandro A.Schïffer, Jinghui Zhang, Zheng Zhang, Webb Miller, and David J. Lipman(1997), “Gapped BLAST and PSI-BLAST: a new generation of proteindatabase search programs”, Nucleic Acids Res. 25:3389-3402.) Other toolsare described herein, specifically in the definition of “Identity.”

Default parameters in the BLAST algorithm include, for example, anexpect threshold of 10, Word size of 28, Match/Mismatch Scores 1, −2,Gap costs Linear. Any filter can be applied as well as a selection forspecies specific repeats, e.g., Homo sapiens.

Peptides

The primary constructs or mmRNA disclosed herein, may encode one or morevalidated or “in testing” proteins or peptides.

According to the present invention, one or more proteins or peptidescurrently being marketed or in development may be encoded by thepolynucleotides, primary constructs or oncology-related mmRNA of thepresent invention. While not wishing to be bound by theory, it isbelieved that incorporation into the primary constructs or mmRNA of theinvention will result in improved therapeutic efficacy due at least inpart to the specificity, purity and selectivity of the constructdesigns.

The polynucleotides, primary constructs and/or mmRNA may alter abiological and/or physiolocial process and/or compound such as, but notlimited to, altering (e.g., slowing) the progression of a disease and/ordisorder, reduce cholesterol and/or low-density lipoprotein (LDL)cholesterol, improve Crigler-Najjar syndrome, restore hepcidin and/orhemochromatosis type 2 function to regulate iron uptake, restore bileacid metabolism, reduce coronary heart disease risk for familialhypercholesterolemia and prevent hyperkeratotic plaques and cornealclouding which may heal hyperkeratotic plaques on the hands and/or feet.

In one embodiment, the polynucleotides, primary constructs and/or mmRNAmay be used to express a polypeptide in cells or tissues for the purposeof replacing the protein produced from a deleted or mutated gene.

Further, the polynucleotides, primary constructs or mmRNA of theinvention may be used to treat metabolic disorders related to rare liverdiseases and/or disorders.

Flanking Regions: Untranslated Regions (UTRs)

Untranslated regions (UTRs) of a gene are transcribed but nottranslated. The 5′UTR starts at the transcription start site andcontinues to the start codon but does not include the start codon;whereas, the 3′UTR starts immediately following the stop codon andcontinues until the transcriptional termination signal. There is growingbody of evidence about the regulatory roles played by the UTRs in termsof stability of the nucleic acid molecule and translation. Theregulatory features of a UTR can be incorporated into thepolynucleotides, primary constructs and/or mmRNA of the presentinvention to enhance the stability of the molecule. The specificfeatures can also be incorporated to ensure controlled down-regulationof the transcript in case they are misdirected to undesired organssites.

5′ UTR and Translation Initiation

Natural 5′UTRs bear features which play roles in for translationinitiation. They harbor signatures like Kozak sequences which arecommonly known to be involved in the process by which the ribosomeinitiates translation of many genes. Kozak sequences have the consensusCCR(A/G)CCAUGG, where R is a purine (adenine or guanine) three basesupstream of the start codon (AUG), which is followed by another ‘G’.5′UTR also have been known to form secondary structures which areinvolved in elongation factor binding.

By engineering the features typically found in abundantly expressedgenes of specific target organs, one can enhance the stability andprotein production of the polynucleotides, primary constructs or mmRNAof the invention. For example, introduction of 5′ UTR of liver-expressedmRNA, such as albumin, serum amyloid A, Apolipoprotein AB/E,transferrin, alpha fetoprotein, erythropoietin, or Factor VIII, could beused to enhance expression of a nucleic acid molecule, such as a mmRNA,in hepatic cell lines or liver. Likewise, use of 5′ UTR from othertissue-specific mRNA to improve expression in that tissue ispossible—for muscle (MyoD, Myosin, Myoglobin, Myogenin, Herculin), forendothelial cells (Tie-1, CD36), for myeloid cells (C/EBP, AML1, G-CSF,GM-CSF, CD11b, MSR, Fr-1, i-NOS), for leukocytes (CD45, CD18), foradipose tissue (CD36, GLUT4, ACRP30, adiponectin) and for lungepithelial cells (SP-A/B/C/D).

Other non-UTR sequences may be incorporated into the 5′ (or 3′ UTR)UTRs. For example, introns or portions of introns sequences may beincorporated into the flanking regions of the polynucleotides, primaryconstructs or mmRNA of the invention. Incorporation of intronicsequences may increase protein production as well as mRNA levels.

The 5′UTR may selected for use in the present invention may be astructured UTR such as, but not limited to, 5′UTRs to controltranslation. As a non-limiting example, a structured 5′UTR may bebeneficial when using any of the terminal modifications described incopending U.S. Provisional Application No. 61/758,921 filed Jan. 31,2013, entitled Differential Targeting Using RNA Constructs; U.S.Provisional Application No. 61/781,139 filed Mar. 14, 2013, entitledDifferential Targeting Using RNA Constructs; U.S. ProvisionalApplication No. 61/729,933, filed Nov. 26, 2012 entitled TerminallyOptimized RNAs; U.S. Provisional Application No. 61/737,224 filed Dec.14, 2012 entitled Terminally Optimized RNAs and U.S. ProvisionalApplication No. 61/829,359 filed May 31, 2013 entitled TerminallyOptimized RNAs; each of which is herein incorporated by reference intheir entirety.

3′ UTR and the AU Rich Elements

3′UTRs are known to have stretches of Adenosines and Uridines embeddedin them. These AU rich signatures are particularly prevalent in geneswith high rates of turnover. Based on their sequence features andfunctional properties, the AU rich elements (AREs) can be separated intothree classes (Chen et al, 1995): Class I AREs contain several dispersedcopies of an AUUUA motif within U-rich regions. C-Myc and MyoD containclass I AREs. Class II AREs possess two or more overlappingUUAUUUA(U/A)(U/A) nonamers. Molecules containing this type of AREsinclude GM-CSF and TNF-α. Class III ARES are less well defined. These Urich regions do not contain an AUUUA motif c-Jun and Myogenin are twowell-studied examples of this class. Most proteins binding to the AREsare known to destabilize the messenger, whereas members of the ELAVfamily, most notably HuR, have been documented to increase the stabilityof mRNA. HuR binds to AREs of all the three classes. Engineering the HuRspecific binding sites into the 3′ UTR of nucleic acid molecules willlead to HuR binding and thus, stabilization of the message in vivo.

Introduction, removal or modification of 3′ UTR AU rich elements (AREs)can be used to modulate the stability of polynucleotides, primaryconstructs or mmRNA of the invention. When engineering specificpolynucleotides, primary constructs or mmRNA, one or more copies of anARE can be introduced to make polynucleotides, primary constructs ormmRNA of the invention less stable and thereby curtail translation anddecrease production of the resultant protein. Likewise, AREs can beidentified and removed or mutated to increase the intracellularstability and thus increase translation and production of the resultantprotein. Transfection experiments can be conducted in relevant celllines, using polynucleotides, primary constructs or mmRNA of theinvention and protein production can be assayed at various time pointspost-transfection. For example, cells can be transfected with differentARE-engineering molecules and by using an ELISA kit to the relevantprotein and assaying protein produced at 6 hr, 12 hr, 24 hr, 48 hr, and7 days post-transfection.

Incorporating microRNA Binding Sites

microRNAs (or miRNA) are 19-25 nucleotide long noncoding RNAs that bindto the 3′UTR of nucleic acid molecules and down-regulate gene expressioneither by reducing nucleic acid molecule stability or by inhibitingtranslation. The polynucleotides, primary constructs or mmRNA of theinvention may comprise one or more microRNA target sequences, microRNAsequences, microRNA binding sites, or microRNA seeds. Such sequences maycorrespond to any known microRNA such as those taught in US PublicationUS2005/0261218 and US Publication US2005/0059005, or those listed inTable 7 of co-pending application U.S. Ser. No. 61/758,921 filed Jan.31, 2013 (Attorney Docket Number 2030.1039), the contents of which areincorporated herein by reference in their entirety.

A microRNA sequence comprises a “seed” region, i.e., a sequence in theregion of positions 2-8 of the mature microRNA, which sequence hasperfect Watson-Crick complementarity to the miRNA target sequence. AmicroRNA seed may comprise positions 2-8 or 2-7 of the mature microRNA.In some embodiments, a microRNA seed may comprise 7 nucleotides (e.g.,nucleotides 2-8 of the mature microRNA), wherein the seed-complementarysite in the corresponding miRNA target is flanked by an adenine (A)opposed to microRNA position 1. In some embodiments, a microRNA seed maycomprise 6 nucleotides (e.g., nucleotides 2-7 of the mature microRNA),wherein the seed-complementary site in the corresponding miRNA target isflanked by an adenine (A) opposed to microRNA position 1. See forexample, Grimson A, Farh K K, Johnston W K, Garrett-Engele P, Lim L P,Bartel D P; Mol Cell. 2007 Jul. 6; 27(1):91-105. The bases of themicroRNA seed have complete complementarity with the target sequence. Byengineering microRNA target sequences into the 3′UTR of polynucleotides,primary constructs or mmRNA of the invention one can target the moleculefor degradation or reduced translation, provided the microRNA inquestion is available. This process will reduce the hazard of off targeteffects upon nucleic acid molecule delivery. Identification of microRNA,microRNA target regions, and their expression patterns and role inbiology have been reported (Bonauer et al., Curr Drug Targets 201011:943-949; Anand and Cheresh Curr Opin Hematol 2011 18:171-176;Contreras and Rao Leukemia 2012 26:404-413 (2011 Dec. 20. doi:10.1038/1eu.2011.356); Bartel Cell 2009 136:215-233; Landgraf et al,Cell, 2007 129:1401-1414).

For example, if the nucleic acid molecule is an mRNA and is not intendedto be delivered to the liver but ends up there, then miR-122, a microRNAabundant in liver, can inhibit the expression of the gene of interest ifone or multiple target sites of miR-122 are engineered into the 3′UTR ofthe polynucleotides, primary constructs or mmRNA. Introduction of one ormultiple binding sites for different microRNA can be engineered tofurther decrease the longevity, stability, and protein translation of apolynucleotides, primary constructs or mmRNA.

As used herein, the term “microRNA site” refers to a microRNA targetsite or a microRNA recognition site, or any nucleotide sequence to whicha microRNA binds or associates. It should be understood that “binding”may follow traditional Watson-Crick hybridization rules or may reflectany stable association of the microRNA with the target sequence at oradjacent to the microRNA site.

Conversely, for the purposes of the polynucleotides, primary constructsor mmRNA of the present invention, microRNA binding sites can beengineered out of (i.e. removed from) sequences in which they naturallyoccur in order to increase protein expression in specific tissues. Forexample, miR-122 binding sites may be removed to improve proteinexpression in the liver. Regulation of expression in multiple tissuescan be accomplished through introduction or removal or one or severalmicroRNA binding sites.

Examples of tissues where microRNA are known to regulate mRNA, andthereby protein expression, include, but are not limited to, liver(miR-122), muscle (miR-133, miR-206, miR-208), endothelial cells(miR-17-92, miR-126), myeloid cells (miR-142-3p, miR-142-5p, miR-16,miR-21, miR-223, miR-24, miR-27), adipose tissue (let-7, miR-30c), heart(miR-1d, miR-149), kidney (miR-192, miR-194, miR-204), and lungepithelial cells (let-7, miR-133, miR-126). MicroRNA can also regulatecomplex biological processes such as angiogenesis (miR-132) (Anand andCheresh Curr Opin Hematol 2011 18:171-176). In the polynucleotides,primary constructs or mmRNA of the invention, binding sites formicroRNAs that are involved in such processes may be removed orintroduced, in order to tailor the expression of the polynucleotides,primary constructs or mmRNA expression to biologically relevant celltypes or to the context of relevant biological processes.

Lastly, through an understanding of the expression patterns of microRNAin different cell types, polynucleotides, primary constructs or mmRNAcan be engineered for more targeted expression in specific cell types oronly under specific biological conditions. Through introduction oftissue-specific microRNA binding sites, polynucleotides, primaryconstructs or mmRNA could be designed that would be optimal for proteinexpression in a tissue or in the context of a biological condition.

Transfection experiments can be conducted in relevant cell lines, usingengineered polynucleotides, primary constructs or mmRNA and proteinproduction can be assayed at various time points post-transfection. Forexample, cells can be transfected with different microRNA bindingsite-engineering polynucleotides, primary constructs or mmRNA and byusing an ELISA kit to the relevant protein and assaying protein producedat 6 hr, 12 hr, 24 hr, 48 hr, 72 hr and 7 days post-transfection. Invivo experiments can also be conducted using microRNA-bindingsite-engineered molecules to examine changes in tissue-specificexpression of formulated polynucleotides, primary constructs or mmRNA.

5′ Capping

The 5′ cap structure of an mRNA is involved in nuclear export,increasing mRNA stability and binds the mRNA Cap Binding Protein (CBP),which is responsible for mRNA stability in the cell and translationcompetency through the association of CBP with poly(A) binding proteinto form the mature cyclic mRNA species. The cap further assists theremoval of 5′ proximal introns removal during mRNA splicing.

Endogenous mRNA molecules may be 5′-end capped generating a5′-ppp-5′-triphosphate linkage between a terminal guanosine cap residueand the 5′-terminal transcribed sense nucleotide of the mRNA molecule.This 5′-guanylate cap may then be methylated to generate anN7-methyl-guanylate residue. The ribose sugars of the terminal and/oranteterminal transcribed nucleotides of the 5′ end of the mRNA mayoptionally also be 2′-O-methylated. 5′-decapping through hydrolysis andcleavage of the guanylate cap structure may target a nucleic acidmolecule, such as an mRNA molecule, for degradation.

Modifications to the polynucleotides, primary constructs, and mmRNA ofthe present invention may generate a non-hydrolyzable cap structurepreventing decapping and thus increasing mRNA half-life. Because capstructure hydrolysis requires cleavage of 5′-ppp-5′ phosphorodiesterlinkages, modified nucleotides may be used during the capping reaction.For example, a Vaccinia Capping Enzyme from New England Biolabs(Ipswich, Mass.) may be used with α-thio-guanosine nucleotides accordingto the manufacturer's instructions to create a phosphorothioate linkagein the 5′-ppp-5′ cap. Additional modified guanosine nucleotides may beused such as α-methyl-phosphonate and seleno-phosphate nucleotides.

Additional modifications include, but are not limited to,2′-O-methylation of the ribose sugars of 5′-terminal and/or5′-anteterminal nucleotides of the mRNA (as mentioned above) on the2′-hydroxyl group of the sugar ring. Multiple distinct 5′-cap structurescan be used to generate the 5′-cap of a nucleic acid molecule, such asan mRNA molecule.

Cap analogs, which herein are also referred to as synthetic cap analogs,chemical caps, chemical cap analogs, or structural or functional capanalogs, differ from natural (i.e. endogenous, wild-type orphysiological) 5′-caps in their chemical structure, while retaining capfunction. Cap analogs may be chemically (i.e. non-enzymatically) orenzymatically synthesized and/linked to a nucleic acid molecule.

For example, the Anti-Reverse Cap Analog (ARCA) cap contains twoguanines linked by a 5′-5′-triphosphate group, wherein one guaninecontains an N7 methyl group as well as a 3′-O-methyl group (i.e.,N7,3′-O-dimethyl-guanosine-5′-triphosphate-5′-guanosine (m⁷G-3′mppp-G;which may equivalently be designated 3′ 0-Me-m7G(5)ppp(5′)G). The 3′-Oatom of the other, unmodified, guanine becomes linked to the 5′-terminalnucleotide of the capped nucleic acid molecule (e.g. an mRNA or mmRNA).The N7- and 3′-O-methlyated guanine provides the terminal moiety of thecapped nucleic acid molecule (e.g. mRNA or mmRNA).

Another exemplary cap is mCAP, which is similar to ARCA but has a2′-O-methyl group on guanosine (i.e.,N7,2′-O-dimethyl-guanosine-5′-triphosphate-5′-guanosine, m⁷Gm-ppp-G).

While cap analogs allow for the concomitant capping of a nucleic acidmolecule in an in vitro transcription reaction, up to 20% of transcriptsremain uncapped. This, as well as the structural differences of a capanalog from an endogenous 5′-cap structures of nucleic acids produced bythe endogenous, cellular transcription machinery, may lead to reducedtranslational competency and reduced cellular stability.

Polynucleotides, primary constructs and mmRNA of the invention may alsobe capped post-transcriptionally, using enzymes, in order to generatemore authentic 5′-cap structures. As used herein, the phrase “moreauthentic” refers to a feature that closely mirrors or mimics, eitherstructurally or functionally, an endogenous or wild type feature. Thatis, a “more authentic” feature is better representative of anendogenous, wild-type, natural or physiological cellular function and/orstructure as compared to synthetic features or analogs, etc., of theprior art, or which outperforms the corresponding endogenous, wild-type,natural or physiological feature in one or more respects. Non-limitingexamples of more authentic 5′cap structures of the present invention arethose which, among other things, have enhanced binding of cap bindingproteins, increased half life, reduced susceptibility to 5′endonucleases and/or reduced 5′decapping, as compared to synthetic 5′capstructures known in the art (or to a wild-type, natural or physiological5′cap structure). For example, recombinant Vaccinia Virus Capping Enzymeand recombinant 2′-O-methyltransferase enzyme can create a canonical5′-5′-triphosphate linkage between the 5′-terminal nucleotide of an mRNAand a guanine cap nucleotide wherein the cap guanine contains an N7methylation and the 5′-terminal nucleotide of the mRNA contains a2′-O-methyl. Such a structure is termed the Cap1 structure. This capresults in a higher translational-competency and cellular stability anda reduced activation of cellular pro-inflammatory cytokines, ascompared, e.g., to other 5′cap analog structures known in the art. Capstructures include 7mG(5′)ppp(5′)N,pN2p (cap 0), 7mG(5′)ppp(5′)NlmpNp(cap 1), and 7mG(5′)-ppp(5′)NlmpN2mp (cap 2).

Because the polynucleotides, primary constructs or mmRNA may be cappedpost-transcriptionally, and because this process is more efficient,nearly 100% of the polynucleotides, primary constructs or mmRNA may becapped. This is in contrast to ˜80% when a cap analog is linked to anmRNA in the course of an in vitro transcription reaction.

According to the present invention, 5′ terminal caps may includeendogenous caps or cap analogs. According to the present invention, a 5′terminal cap may comprise a guanine analog. Useful guanine analogsinclude inosine, N1-methyl-guanosine, 2′fluoro-guanosine,7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine,and 2-azido-guanosine.

Viral Sequences

Additional viral sequences such as, but not limited to, the translationenhancer sequence of the barley yellow dwarf virus (BYDV-PAV) can beengineered and inserted in the 3′ UTR of the polynucleotides, primaryconstructs or mmRNA of the invention and can stimulate the translationof the construct in vitro and in vivo. Transfection experiments can beconducted in relevant cell lines at and protein production can beassayed by ELISA at 12 hr, 24 hr, 48 hr, 72 hr and day 7post-transfection.

IRES Sequences

Further, provided are polynucleotides, primary constructs or mmRNA whichmay contain an internal ribosome entry site (IRES). First identified asa feature Picorna virus RNA, IRES plays an important role in initiatingprotein synthesis in absence of the 5′ cap structure. An IRES may act asthe sole ribosome binding site, or may serve as one of multiple ribosomebinding sites of an mRNA. polynucleotides, primary constructs or mmRNAcontaining more than one functional ribosome binding site may encodeseveral peptides or polypeptides that are translated independently bythe ribosomes (“multicistronic nucleic acid molecules”). Whenpolynucleotides, primary constructs or mmRNA are provided with an IRES,further optionally provided is a second translatable region. Examples ofIRES sequences that can be used according to the invention includewithout limitation, those from picornaviruses (e.g. FMDV), pest viruses(CFFV), polio viruses (PV), encephalomyocarditis viruses (ECMV),foot-and-mouth disease viruses (FMDV), hepatitis C viruses (HCV),classical swine fever viruses (CSFV), murine leukemia virus (MLV),simian immune deficiency viruses (SIV) or cricket paralysis viruses(CrPV).

Poly-A Tails

During RNA processing, a long chain of adenine nucleotides (poly-A tail)may be added to a polynucleotide such as an mRNA molecules in order toincrease stability. Immediately after transcription, the 3′ end of thetranscript may be cleaved to free a 3′ hydroxyl. Then poly-A polymeraseadds a chain of adenine nucleotides to the RNA. The process, calledpolyadenylation, adds a poly-A tail that can be between 100 and 250residues long.

It has been discovered that unique poly-A tail lengths provide certainadvantages to the polynucleotides, primary constructs or mmRNA of thepresent invention.

Generally, the length of a poly-A tail of the present invention isgreater than 30 nucleotides in length. In another embodiment, the poly-Atail is greater than 35 nucleotides in length (e.g., at least or greaterthan about 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180,200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,100,1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,500,and 3,000 nucleotides). In some embodiments, the polynucleotide, primaryconstruct, or mmRNA includes from about 30 to about 3,000 nucleotides(e.g., from 30 to 50, from 30 to 100, from 30 to 250, from 30 to 500,from 30 to 750, from 30 to 1,000, from 30 to 1,500, from 30 to 2,000,from 30 to 2,500, from 50 to 100, from 50 to 250, from 50 to 500, from50 to 750, from 50 to 1,000, from 50 to 1,500, from 50 to 2,000, from 50to 2,500, from 50 to 3,000, from 100 to 500, from 100 to 750, from 100to 1,000, from 100 to 1,500, from 100 to 2,000, from 100 to 2,500, from100 to 3,000, from 500 to 750, from 500 to 1,000, from 500 to 1,500,from 500 to 2,000, from 500 to 2,500, from 500 to 3,000, from 1,000 to1,500, from 1,000 to 2,000, from 1,000 to 2,500, from 1,000 to 3,000,from 1,500 to 2,000, from 1,500 to 2,500, from 1,500 to 3,000, from2,000 to 3,000, from 2,000 to 2,500, and from 2,500 to 3,000).

In one embodiment, the poly-A tail is designed relative to the length ofthe overall polynucleotides, primary constructs or mmRNA. This designmay be based on the length of the coding region, the length of aparticular feature or region (such as the first or flanking regions), orbased on the length of the ultimate product expressed from thepolynucleotides, primary constructs or mmRNA.

In this context the poly-A tail may be 10, 20, 30, 40, 50, 60, 70, 80,90, or 100% greater in length than the polynucleotides, primaryconstructs or mmRNA or feature thereof. The poly-A tail may also bedesigned as a fraction of polynucleotides, primary constructs or mmRNAto which it belongs. In this context, the poly-A tail may be 10, 20, 30,40, 50, 60, 70, 80, or 90% or more of the total length of the constructor the total length of the construct minus the poly-A tail. Further,engineered binding sites and conjugation of polynucleotides, primaryconstructs or mmRNA for Poly-A binding protein may enhance expression.

Additionally, multiple distinct polynucleotides, primary constructs ormmRNA may be linked together to the PABP (Poly-A binding protein)through the 3′-end using modified nucleotides at the 3′-terminus of thepoly-A tail. Transfection experiments can be conducted in relevant celllines at and protein production can be assayed by ELISA at 12 hr, 24 hr,48 hr, 72 hr and day 7 post-transfection.

In one embodiment, the polynucleotide primary constructs of the presentinvention are designed to include a polyA-G Quartet. The G-quartet is acyclic hydrogen bonded array of four guanine nucleotides that can beformed by G-rich sequences in both DNA and RNA. In this embodiment, theG-quartet is incorporated at the end of the poly-A tail. The resultantmmRNA construct is assayed for stability, protein production and otherparameters including half-life at various time points. It has beendiscovered that the polyA-G quartet results in protein productionequivalent to at least 75% of that seen using a poly-A tail of 120nucleotides alone.

Quantification

In one embodiment, the polynucleotides, primary constructs or mmRNA ofthe present invention may be quantified in exosomes derived from one ormore bodily fluid. As used herein “bodily fluids” include peripheralblood, serum, plasma, ascites, urine, cerebrospinal fluid (CSF), sputum,saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid,cerumen, breast milk, broncheoalveolar lavage fluid, semen, prostaticfluid, cowper's fluid or pre-ejaculatory fluid, sweat, fecal matter,hair, tears, cyst fluid, pleural and peritoneal fluid, pericardialfluid, lymph, chyme, chyle, bile, interstitial fluid, menses, pus,sebum, vomit, vaginal secretions, mucosal secretion, stool water,pancreatic juice, lavage fluids from sinus cavities, bronchopulmonaryaspirates, blastocyl cavity fluid, and umbilical cord blood.Alternatively, exosomes may be retrieved from an organ selected from thegroup consisting of lung, heart, pancreas, stomach, intestine, bladder,kidney, ovary, testis, skin, colon, breast, prostate, brain, esophagus,liver, and placenta.

In the quantification method, a sample of not more than 2 mL is obtainedfrom the subject and the exosomes isolated by size exclusionchromatography, density gradient centrifugation, differentialcentrifugation, nanomembrane ultrafiltration, immunoabsorbent capture,affinity purification, microfluidic separation, or combinations thereof.In the analysis, the level or concentration of a polynucleotide, primaryconstruct or mmRNA may be an expression level, presence, absence,truncation or alteration of the administered construct. It isadvantageous to correlate the level with one or more clinical phenotypesor with an assay for a human disease biomarker. The assay may beperformed using construct specific probes, cytometry, qRT-PCR, real-timePCR, PCR, flow cytometry, electrophoresis, mass spectrometry, orcombinations thereof while the exosomes may be isolated usingimmunohistochemical methods such as enzyme linked immunosorbent assay(ELISA) methods. Exosomes may also be isolated by size exclusionchromatography, density gradient centrifugation, differentialcentrifugation, nanomembrane ultrafiltration, immunoabsorbent capture,affinity purification, microfluidic separation, or combinations thereof.

These methods afford the investigator the ability to monitor, in realtime, the level of polynucleotides, primary constructs or mmRNAremaining or delivered. This is possible because the polynucleotides,primary constructs or mmRNA of the present invention differ from theendogenous forms due to the structural or chemical modifications.

II. Design and Synthesis of mmRNA

Polynucleotides, primary constructs or mmRNA for use in accordance withthe invention may be prepared according to any available techniqueincluding, but not limited to chemical synthesis, enzymatic synthesis,which is generally termed in vitro transcription (IVT) or enzymatic orchemical cleavage of a longer precursor, etc. Methods of synthesizingRNAs are known in the art (see, e.g., Gait, M. J. (ed.) Oligonucleotidesynthesis: a practical approach, Oxford [Oxfordshire], Washington, DC:IRL Press, 1984; and Herdewijn, P. (ed.) Oligonucleotide synthesis:methods and applications, Methods in Molecular Biology, v. 288 (Clifton,N.J.) Totowa, N.J.: Humana Press, 2005; both of which are incorporatedherein by reference).

The process of design and synthesis of the primary constructs of theinvention generally includes the steps of gene construction, mRNAproduction (either with or without modifications) and purification. Inthe enzymatic synthesis method, a target polynucleotide sequenceencoding the polypeptide of interest is first selected for incorporationinto a vector which will be amplified to produce a cDNA template.Optionally, the target polynucleotide sequence and/or any flankingsequences may be codon optimized. The cDNA template is then used toproduce mRNA through in vitro transcription (IVT). After production, themRNA may undergo purification and clean-up processes. The steps of whichare provided in more detail below.

Gene Construction

The step of gene construction may include, but is not limited to genesynthesis, vector amplification, plasmid purification, plasmidlinearization and clean-up, and cDNA template synthesis and clean-up.

Gene Synthesis

Once a polypeptide of interest, or target, is selected for production, aprimary construct is designed. Within the primary construct, a firstregion of linked nucleosides encoding the polypeptide of interest may beconstructed using an open reading frame (ORF) of a selected nucleic acid(DNA or RNA) transcript. The ORF may comprise the wild type ORF, anisoform, variant or a fragment thereof. As used herein, an “open readingframe” or “ORF” is meant to refer to a nucleic acid sequence (DNA orRNA) which is capable of encoding a polypeptide of interest. ORFs oftenbegin with the start codon, ATG and end with a nonsense or terminationcodon or signal.

Further, the nucleotide sequence of the first region may be codonoptimized. Codon optimization methods are known in the art and may beuseful in efforts to achieve one or more of several goals. These goalsinclude to match codon frequencies in target and host organisms toensure proper folding, bias GC content to increase mRNA stability orreduce secondary structures, minimize tandem repeat codons or base runsthat may impair gene construction or expression, customizetranscriptional and translational control regions, insert or removeprotein trafficking sequences, remove/add post translation modificationsites in encoded protein (e.g. glycosylation sites), add, remove orshuffle protein domains, insert or delete restriction sites, modifyribosome binding sites and mRNA degradation sites, to adjusttranslational rates to allow the various domains of the protein to foldproperly, or to reduce or eliminate problem secondary structures withinthe mRNA. Codon optimization tools, algorithms and services are known inthe art, non-limiting examples include services from GeneArt (LifeTechnologies) and/or DNA2.0 (Menlo Park Calif.). In one embodiment, theORF sequence is optimized using optimization algorithms. Codon optionsfor each amino acid are given in Table 1.

TABLE 1 Codon Options Single Letter Amino Acid Code Codon OptionsIsoleucine I ATT, ATC, ATA Leucine L CTT, CTC, CTA, CTG, TTA, TTG ValineV GTT, GTC, GTA, GTG Phenylalanine F TTT, TTC Methionine M ATG CysteineC TGT, TGC Alanine A GCT, GCC, GCA, GCG Glycine G GGT, GGC, GGA, GGGProline P CCT, CCC, CCA, CCG Threonine T ACT, ACC, ACA, ACG Serine STCT, TCC, TCA, TCG, AGT, AGC Tyrosine Y TAT, TAC Tryptophan W TGGGlutamine Q CAA, CAG Asparagine N AAT, AAC Histidine H CAT, CAC Glutamicacid E GAA, GAG Aspartic acid D GAT, GAC Lysine K AAA, AAG Arginine RCGT, CGC, CGA, CGG, AGA, AGG Selenocysteine Sec UGA in mRNA in presenceof Selenocystein insertion element (SECIS) Stop codons Stop TAA, TAG,TGA

In one embodiment, at least a portion of the modified mRNA nucleotidesequence may be codon optimized by methods known in the art and/ordescribed herein. After a sequence has been codon optimized it may befurther evaluated for regions containing restriction sites. At least onenucleotide within the restriction site regions may be replaced withanother nucleotide in order to remove the restriction site from thesequence but the replacement of nucleotides does alter the amino acidsequence which is encoded by the codon optimized nucleotide sequence.

Features, which may be considered beneficial in some embodiments of thepresent invention, may be encoded by the primary construct and may flankthe ORF as a first or second flanking region. The flanking regions maybe incorporated into the primary construct before and/or afteroptimization of the ORF. It is not required that a primary constructcontain both a 5′ and 3′ flanking region. Examples of such featuresinclude, but are not limited to, untranslated regions (UTRs), Kozaksequences, an oligo(dT) sequence, and detectable tags and may includemultiple cloning sites which may have XbaI recognition.

In some embodiments, a 5′ UTR and/or a 3′ UTR may be provided asflanking regions. Multiple 5′ or 3′ UTRs may be included in the flankingregions and may be the same or of different sequences. Any portion ofthe flanking regions, including none, may be codon optimized and any mayindependently contain one or more different structural or chemicalmodifications, before and/or after codon optimization. Combinations offeatures may be included in the first and second flanking regions andmay be contained within other features. For example, the ORF may beflanked by a 5′ UTR which may contain a strong Kozak translationalinitiation signal and/or a 3′ UTR which may include an oligo(dT)sequence for templated addition of a poly-A tail.

Tables 2 and 3 of co-pending U.S. Provisional Patent Application No.61/737,130 filed Dec. 14, 2012 provide a listing of exemplary UTRs whichmay be utilized in the primary construct of the present invention asflanking regions. Variants of 5′ or 3′UTRs may be utilized wherein oneor more nucleotides are added or removed to the termini, including A, T,C or G.

It should be understood that those listed are examples and that any UTRfrom any gene may be incorporated into the respective first or secondflanking region of the primary construct. Furthermore, multiplewild-type UTRs of any known gene may be utilized. It is also within thescope of the present invention to provide artificial UTRs which are notvariants of wild type genes. These UTRs or portions thereof may beplaced in the same orientation as in the transcript from which they wereselected or may be altered in orientation or location. Hence a 5′ or 3′UTR may be inverted, shortened, lengthened, made chimeric with one ormore other 5′ UTRs or 3′ UTRs. As used herein, the term “altered” as itrelates to a UTR sequence, means that the UTR has been changed in someway in relation to a reference sequence. For example, a 3′ or 5′ UTR maybe altered relative to a wild type or native UTR by the change inorientation or location as taught above or may be altered by theinclusion of additional nucleotides, deletion of nucleotides, swappingor transposition of nucleotides. Any of these changes producing an“altered” UTR (whether 3′ or 5′) comprise a variant UTR.

In one embodiment, a double, triple or quadruple UTR such as a 5′ or 3′UTR may be used. As used herein, a “double” UTR is one in which twocopies of the same UTR are encoded either in series or substantially inseries. For example, a double beta-globin 3′ UTR may be used asdescribed in US Patent publication 20100129877, the contents of whichare incorporated herein by reference in its entirety.

It is also within the scope of the present invention to have patternedUTRs. As used herein “patterned UTRs” are those UTRs which reflect arepeating or alternating pattern, such as ABABAB or AABBAABBAABB orABCABCABC or variants thereof repeated once, twice, or more than 3times. In these patterns, each letter, A, B, or C represent a differentUTR at the nucleotide level.

In one embodiment, flanking regions are selected from a family oftranscripts whose proteins share a common function, structure, featureof property. For example, polypeptides of interest may belong to afamily of proteins which are expressed in a particular cell, tissue orat some time during development. The UTRs from any of these genes may beswapped for any other UTR of the same or different family of proteins tocreate a new chimeric primary transcript. As used herein, a “family ofproteins” is used in the broadest sense to refer to a group of two ormore polypeptides of interest which share at least one function,structure, feature, localization, origin, or expression pattern.

After optimization (if desired), the primary construct components arereconstituted and transformed into a vector such as, but not limited to,plasmids, viruses, cosmids, and artificial chromosomes. For example, theoptimized construct may be reconstituted and transformed into chemicallycompetent E. coli, yeast, neurospora, maize, drosophila, etc. where highcopy plasmid-like or chromosome structures occur by methods describedherein.

Stop Codons

In one embodiment, the primary constructs of the present invention mayinclude at least two stop codons before the 3′ untranslated region(UTR). The stop codon may be selected from TGA, TAA and TAG. In oneembodiment, the primary constructs of the present invention include thestop codon TGA and one additional stop codon. In a further embodimentthe addition stop codon may be TAA.

In another embodiment, the primary constructs of the present inventionmay include three stop codons before the 3′ untranslated region (UTR).

Vector Amplification

The vector containing the primary construct is then amplified and theplasmid isolated and purified using methods known in the art such as,but not limited to, a maxi prep using the Invitrogen PURELINK™ HiPureMaxiprep Kit (Carlsbad, Calif.).

Plasmid Linearization

The plasmid may then be linearized using methods known in the art suchas, but not limited to, the use of restriction enzymes and buffers. Thelinearization reaction may be purified using methods including, forexample Invitrogen's PURELINK™ PCR Micro Kit (Carlsbad, Calif.), andHPLC based purification methods such as, but not limited to, stronganion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC(RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC) and Invitrogen'sstandard PURELINK™ PCR Kit (Carlsbad, Calif.). The purification methodmay be modified depending on the size of the linearization reactionwhich was conducted. The linearized plasmid is then used to generatecDNA for in vitro transcription (IVT) reactions.

cDNA Template Synthesis

A cDNA template may be synthesized by having a linearized plasmidundergo polymerase chain reaction (PCR). Table 4 of U.S. ProvisionalPatent Application No. 61/737,130 filed Dec. 14, 2012 provides a listingof primers and probes that may be usefully in the PCR reactions of thepresent invention. It should be understood that the listing is notexhaustive and that primer-probe design for any amplification is withinthe skill of those in the art. Probes may also contain chemicallymodified bases to increase base-pairing fidelity to the target moleculeand base-pairing strength.

In one embodiment, the cDNA may be submitted for sequencing analysisbefore undergoing transcription.

mRNA Production

The process of mRNA or mmRNA production may include, but is not limitedto, in vitro transcription, cDNA template removal and RNA clean-up, andmRNA capping and/or tailing reactions.

In Vitro Transcription

The cDNA produced in the previous step may be transcribed using an invitro transcription (IVT) system. The system typically comprises atranscription buffer, nucleotide triphosphates (NTPs), an RNaseinhibitor and a polymerase. The NTPs may be manufactured in house, maybe selected from a supplier, or may be synthesized as described herein.The NTPs may be selected from, but are not limited to, those describedherein including natural and unnatural (modified) NTPs. The polymerasemay be selected from, but is not limited to, T7 RNA polymerase, T3 RNApolymerase and mutant polymerases such as, but not limited to,polymerases able to incorporate modified nucleic acids.

RNA Polymerases

Any number of RNA polymerases or variants may be used in the design ofthe primary constructs of the present invention.

RNA polymerases may be modified by inserting or deleting amino acids ofthe RNA polymerase sequence. As a non-limiting example, the RNApolymerase may be modified to exhibit an increased ability toincorporate a 2′-modified nucleotide triphosphate compared to anunmodified RNA polymerase (see International Publication WO2008078180and U.S. Pat. No. 8,101,385; herein incorporated by reference in theirentireties).

Variants may be obtained by evolving an RNA polymerase, optimizing theRNA polymerase amino acid and/or nucleic acid sequence and/or by usingother methods known in the art. As a non-limiting example, T7 RNApolymerase variants may be evolved using the continuous directedevolution system set out by Esvelt et al. (Nature (2011)472(7344):499-503; herein incorporated by reference in its entirety)where clones of T7 RNA polymerase may encode at least one mutation suchas, but not limited to, lysine at position 93 substituted for threonine(K93T), I4M, A7T, E63V, V64D, A65E, D66Y, T76N, C125R, S128R, A136T,N165S, G175R, H176L, Y178H, F182L, L196F, G198V, D208Y, E222K, S228A,Q239R, T243N, G259D, M267I, G280C, H300R, D351A, A354S, E356D, L360P,A383V, Y385C, D388Y, S397R, M401T, N410S, K450R, P451T, G452V, E484A,H523L, H524N, G542V, E565K, K577E, K577M, N601S, S684Y, L699I, K713E,N748D, Q754R, E775K, A827V, D851N or L864F. As another non-limitingexample, T7 RNA polymerase variants may encode at least mutation asdescribed in U.S. Pub. Nos. 20100120024 and 20070117112; hereinincorporated by reference in their entireties. Variants of RNApolymerase may also include, but are not limited to, substitutionalvariants, conservative amino acid substitution, insertional variants,deletional variants and/or covalent derivatives.

In one embodiment, the primary construct may be designed to berecognized by the wild type or variant RNA polymerases. In doing so, theprimary construct may be modified to contain sites or regions ofsequence changes from the wild type or parent primary construct.

In one embodiment, the primary construct may be designed to include atleast one substitution and/or insertion upstream of an RNA polymerasebinding or recognition site, downstream of the RNA polymerase binding orrecognition site, upstream of the TATA box sequence, downstream of theTATA box sequence of the primary construct but upstream of the codingregion of the primary construct, within the 5′UTR, before the 5′UTRand/or after the 5′UTR.

In one embodiment, the 5′UTR of the primary construct may be replaced bythe insertion of at least one region and/or string of nucleotides of thesame base. The region and/or string of nucleotides may include, but isnot limited to, at least 3, at least 4, at least 5, at least 6, at least7 or at least 8 nucleotides and the nucleotides may be natural and/orunnatural. As a non-limiting example, the group of nucleotides mayinclude 5-8 adenine, cytosine, thymine, a string of any of the othernucleotides disclosed herein and/or combinations thereof.

In one embodiment, the 5′UTR of the primary construct may be replaced bythe insertion of at least two regions and/or strings of nucleotides oftwo different bases such as, but not limited to, adenine, cytosine,thymine, any of the other nucleotides disclosed herein and/orcombinations thereof. For example, the 5′UTR may be replaced byinserting 5-8 adenine bases followed by the insertion of 5-8 cytosinebases. In another example, the 5′UTR may be replaced by inserting 5-8cytosine bases followed by the insertion of 5-8 adenine bases.

In one embodiment, the primary construct may include at least onesubstitution and/or insertion downstream of the transcription start sitewhich may be recognized by an RNA polymerase. As a non-limiting example,at least one substitution and/or insertion may occur downstream thetranscription start site by substituting at least one nucleic acid inthe region just downstream of the transcription start site (such as, butnot limited to, +1 to +6). Changes to region of nucleotides justdownstream of the transcription start site may affect initiation rates,increase apparent nucleotide triphosphate (NTP) reaction constantvalues, and increase the dissociation of short transcripts from thetranscription complex curing initial transcription (Brieba et al,Biochemistry (2002) 41: 5144-5149; herein incorporated by reference inits entirety). The modification, substitution and/or insertion of atleast one nucleic acid may cause a silent mutation of the nucleic acidsequence or may cause a mutation in the amino acid sequence.

In one embodiment, the primary construct may include the substitution ofat least 1, at least 2, at least 3, at least 4, at least 5, at least 6,at least 7, at least 8, at least 9, at least 10, at least 11, at least12 or at least 13 guanine bases downstream of the transcription startsite.

In one embodiment, the primary construct may include the substitution ofat least 1, at least 2, at least 3, at least 4, at least 5 or at least 6guanine bases in the region just downstream of the transcription startsite. As a non-limiting example, if the nucleotides in the region areGGGAGA the guanine bases may be substituted by at least 1, at least 2,at least 3 or at least 4 adenine nucleotides. In another non-limitingexample, if the nucleotides in the region are GGGAGA the guanine basesmay be substituted by at least 1, at least 2, at least 3 or at least 4cytosine bases. In another non-limiting example, if the nucleotides inthe region are GGGAGA the guanine bases may be substituted by at least1, at least 2, at least 3 or at least 4 thymine, and/or any of thenucleotides described herein.

In one embodiment, the primary construct may include at least onesubstitution and/or insertion upstream of the start codon. For thepurpose of clarity, one of skill in the art would appreciate that thestart codon is the first codon of the protein coding region whereas thetranscription start site is the site where transcription begins. Theprimary construct may include, but is not limited to, at least 1, atleast 2, at least 3, at least 4, at least 5, at least 6, at least 7 orat least 8 substitutions and/or insertions of nucleotide bases. Thenucleotide bases may be inserted or substituted at 1, at least 1, atleast 2, at least 3, at least 4 or at least 5 locations upstream of thestart codon. The nucleotides inserted and/or substituted may be the samebase (e.g., all A or all C or all T or all G), two different bases(e.g., A and C, A and T, or C and T), three different bases (e.g., A, Cand T or A, C and T) or at least four different bases. As a non-limitingexample, the guanine base upstream of the coding region in the primaryconstruct may be substituted with adenine, cytosine, thymine, or any ofthe nucleotides described herein. In another non-limiting example thesubstitution of guanine bases in the primary construct may be designedso as to leave one guanine base in the region downstream of thetranscription start site and before the start codon (see Esvelt et al.Nature (2011) 472(7344):499-503; herein incorporated by reference in itsentirety). As a non-limiting example, at least 5 nucleotides may beinserted at 1 location downstream of the transcription start site butupstream of the start codon and the at least 5 nucleotides may be thesame base type.

cDNA Template Removal and Clean-Up

The cDNA template may be removed using methods known in the art such as,but not limited to, treatment with Deoxyribonuclease I (DNase I). RNAclean-up may also include a purification method such as, but not limitedto, AGENCOURT® CLEANSEQ® system from Beckman Coulter (Danvers, Mass.),HPLC based purification methods such as, but not limited to, stronganion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC(RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC).

Capping and/or Tailing Reactions

The primary construct or mmRNA may also undergo capping and/or tailingreactions. A capping reaction may be performed by methods known in theart to add a 5′ cap to the 5′ end of the primary construct. Methods forcapping include, but are not limited to, using a Vaccinia Capping enzyme(New England Biolabs, Ipswich, Mass.).

A poly-A tailing reaction may be performed by methods known in the art,such as, but not limited to, 2′ O-methyltransferase and by methods asdescribed herein. If the primary construct generated from cDNA does notinclude a poly-T, it may be beneficial to perform the poly-A-tailingreaction before the primary construct is cleaned.

mRNA Purification

Primary construct or mmRNA purification may include, but is not limitedto, mRNA or mmRNA clean-up, quality assurance and quality control. mRNAor mmRNA clean-up may be performed by methods known in the arts such as,but not limited to, AGENCOURT® beads (Beckman Coulter Genomics, Danvers,Mass.), poly-T beads, LNA™ oligo-T capture probes (EXIQON® Inc, Vedbaek,Denmark) or HPLC based purification methods such as, but not limited to,strong anion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC(RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC). The term“purified” when used in relation to a polynucleotide such as a “purifiedmRNA or mmRNA” refers to one that is separated from at least onecontaminant. As used herein, a “contaminant” is any substance whichmakes another unfit, impure or inferior. Thus, a purified polynucleotide(e.g., DNA and RNA) is present in a form or setting different from thatin which it is found in nature, or a form or setting different from thatwhich existed prior to subjecting it to a treatment or purificationmethod.

A quality assurance and/or quality control check may be conducted usingmethods such as, but not limited to, gel electrophoresis, UV absorbance,or analytical HPLC.

In another embodiment, the mRNA or mmRNA may be sequenced by methodsincluding, but not limited to reverse-transcriptase-PCR.

In one embodiment, the mRNA or mmRNA may be quantified using methodssuch as, but not limited to, ultraviolet visible spectroscopy (UV/Vis).A non-limiting example of a UV/Vis spectrometer is a NANODROP®spectrometer (ThermoFisher, Waltham, Mass.). The quantified mRNA ormmRNA may be analyzed in order to determine if the mRNA or mmRNA may beof proper size, check that no degradation of the mRNA or mmRNA hasoccurred. Degradation of the mRNA and/or mmRNA may be checked by methodssuch as, but not limited to, agarose gel electrophoresis, HPLC basedpurification methods such as, but not limited to, strong anion exchangeHPLC, weak anion exchange HPLC, reverse phase HPLC (RP-HPLC), andhydrophobic interaction HPLC (HIC-HPLC), liquid chromatography-massspectrometry (LCMS), capillary electrophoresis (CE) and capillary gelelectrophoresis (CGE).

Signal Sequences

The primary constructs or mmRNA may also encode additional featureswhich facilitate trafficking of the polypeptides to therapeuticallyrelevant sites. One such feature which aids in protein trafficking isthe signal sequence. As used herein, a “signal sequence” or “signalpeptide” is a polynucleotide or polypeptide, respectively, which is fromabout 9 to 200 nucleotides (3-60 amino acids) in length which isincorporated at the 5′ (or N-terminus) of the coding region orpolypeptide encoded, respectively. Addition of these sequences result intrafficking of the encoded polypeptide to the endoplasmic reticulumthrough one or more secretory pathways. Some signal peptides are cleavedfrom the protein by signal peptidase after the proteins are transported.

Signal sequences may be selected from any of those listed in co-pendingU.S. Provisional Patent Application No. 61/737,130 filed Dec. 14, 2012,the contents of which are incorporated herein by reference. Proteinsignal sequences which may be incorporated for encoding by thepolynucleotides, primary constructs or mmRNA of the invention includesignal sequences from α-1-antitrypsin, G-CSF, Factor IX, Prolactin,Albumin, HMMSP38, ornithine carbamoyltransferase, Cytochrome C Oxidasesubunit 8A, Type III, bacterial, viral, secretion signals, Vrg-6, PhoA,OmpA, STI, STII, Amylase, Alpha Factor, Endoglucanase V, Secretionsignal, fungal and fibronectin.

In the table, SS is secretion signal and MLS is mitochondrial leadersignal. The primary constructs or mmRNA of the present invention may bedesigned to encode any of the signal sequences or fragments or variantsthereof. These sequences may be included at the beginning of thepolypeptide coding region, in the middle or at the terminus oralternatively into a flanking region.

Additional signal sequences which may be utilized in the presentinvention include those taught in, for example, databases such as thosefound at http://www.signalpeptide.de/ orhttp://proline.bic.nus.edu.sg/spdb/. Those described in U.S. Pat. Nos.8,124,379; 7,413,875 and 7,385,034 are also within the scope of theinvention and the contents of each are incorporated herein by referencein their entirety.

Target Selection

According to the present invention, the primary constructs comprise atleast a first region of linked nucleosides encoding at least onepolypeptide of interest. The polypeptides of interest or “Targets” ofthe present invention are listed in Table 2 below. Shown in Table 2, inaddition to the name and description of the gene encoding thepolypeptide of interest are the ENSEMBL Transcript ID (ENST), theENSEMBL Protein ID (ENSP) and when available the optimized sequence ID(OPT SEQ ID). For any particular gene there may exist one or morevariants or isoforms. Where these exist, they are shown in the table aswell. It will be appreciated by those of skill in the art that disclosedin the Table are potential flanking regions. These are encoded in eachENST transcript either to the 5′ (upstream) or 3′ (downstream) of theORF or coding region. The coding region is definitively and specificallydisclosed by teaching the ENSP sequence. Consequently, the sequencestaught flanking that encoding the protein are considered flankingregions. It is also possible to further characterize the 5′ and 3′flanking regions by utilizing one or more available databases oralgorithms. Databases have annotated the features contained in theflanking regions of the ENST transcripts and these are available in theart.

TABLE 2 Targets Transcript Protein Target Gene Description ENST SEQ IDNO ENSP SEQ ID NO 1 LDLR low density lipoprotein 455727 1 397829 17receptor 2 LDLR low density lipoprotein 561343 2 454147 18 receptor 3LDLR low density lipoprotein 558518 3 454071 19 receptor 4 LDLR lowdensity lipoprotein 558013 4 453346 20 receptor 5 LDLR low densitylipoprotein 535915 5 440520 21 receptor 6 LDLR low density lipoprotein545707 6 437639 22 receptor 7 LDLR1_D331E low density lipoprotein none 7none — PCSK9 mutant receptor/PCSK9 mutant 8 LDLR1_L339D low densitylipoprotein none 8 none — PCSK9 mutant receptor/PCSK9 mutant 9LDLR1_N316A low density lipoprotein none 9 none — PCSK9 Mutantreceptor/PCSK9 mutant 10 LDLR1_E317A low density lipoprotein none 10none — PCSK9 Mutant receptor/PCSK9 mutant 11 LDLR1_Y336A low densitylipoprotein none 11 none — PCSK9 Mutant receptor/PCSK9 mutant 12LDLR1_4A low density lipoprotein none 12 none — PCSK9 mutantreceptor/PCSK9 mutant 13 CYP7A1 Cholesterol 7alpha none 13 (combine none23 hydroxylase SEQ ID NO 39, 40 and 41) 14 PCSK9 proprotein convertase543384 14 441859 24 subtilisin/kexin type 9 15 PCSK9 proproteinconvertase 452118 15 401598 25 subtilisin/kexin type 9 16 PCSK9proprotein convertase 302118 16 303208 26 subtilisin/kexin type 9

In one embodiment, the targets of the present invention may be any ofthe targets described in U.S. Provisional Patent Application No.61/618,862, filed Apr. 2, 2012, entitled Modified Polynucleotides forthe Production of Biologics; U.S. Provisional Patent Application No.61/681,645, filed Aug. 10, 2012, entitled Modified Polynucleotides forthe Production of Biologics; U.S. Provisional Patent Application No.61/737,130, filed Dec. 14, 2012, entitled Modified Polynucleotides forthe Production of Biologics; U.S. Provisional Patent Application No.61/618,866, filed Apr. 2, 2012, entitled Modified Polynucleotides forthe Production of Antibodies; U.S. Provisional Patent Application No.61/681,647, filed Aug. 10, 2012, entitled Modified Polynucleotides forthe Production of Antibodies; U.S. Provisional Patent Application No.61/737,134, filed Dec. 14, 2012, entitled Modified Polynucleotides forthe Production of Antibodies; U.S. Provisional Patent Application No.61/618,868, filed Apr. 2, 2012, entitled Modified Polynucleotides forthe Production of Vaccines; U.S. Provisional Patent Application No.61/681,648, filed Aug. 10, 2012, entitled Modified Polynucleotides forthe Production of Vaccines; U.S. Provisional Patent Application No.61/737,135, filed Dec. 14, 2012, entitled Modified Polynucleotides forthe Production of Vaccines; U.S. Provisional Patent Application No.61/618,870, filed Apr. 2, 2012, entitled Modified Polynucleotides forthe Production of Therapeutic Proteins and Peptides; U.S. ProvisionalPatent Application No. 61/681,649, filed Aug. 10, 2012, entitledModified Polynucleotides for the Production of Therapeutic Proteins andPeptides; U.S. Provisional Patent Application No. 61/737,139, filed Dec.14, 2012, Modified Polynucleotides for the Production of TherapeuticProteins and Peptides; U.S. Provisional Patent Application No.61/618,873, filed Apr. 2, 2012, entitled Modified Polynucleotides forthe Production of Secreted Proteins; U.S. Provisional Patent ApplicationNo. 61/681,650, filed Aug. 10, 2012, entitled Modified Polynucleotidesfor the Production of Secreted Proteins; U.S. Provisional PatentApplication No. 61/737,147, filed Dec. 14, 2012, entitled ModifiedPolynucleotides for the Production of Secreted Proteins; U.S.Provisional Patent Application No. 61/618,878, filed Apr. 2, 2012,entitled Modified Polynucleotides for the Production of Plasma MembraneProteins; U.S. Provisional Patent Application No. 61/681,654, filed Aug.10, 2012, entitled Modified Polynucleotides for the Production of PlasmaMembrane Proteins; U.S. Provisional Patent Application No. 61/737,152,filed Dec. 14, 2012, entitled Modified Polynucleotides for theProduction of Plasma Membrane Proteins; U.S. Provisional PatentApplication No. 61/618,885, filed Apr. 2, 2012, entitled ModifiedPolynucleotides for the Production of Cytoplasmic and CytoskeletalProteins; U.S. Provisional Patent Application No. 61/681,658, filed Aug.10, 2012, entitled Modified Polynucleotides for the Production ofCytoplasmic and Cytoskeletal Proteins; U.S. Provisional PatentApplication No. 61/737,155, filed Dec. 14, 2012, entitled ModifiedPolynucleotides for the Production of Cytoplasmic and CytoskeletalProteins; U.S. Provisional Patent Application No. 61/618,896, filed Apr.2, 2012, entitled Modified Polynucleotides for the Production ofIntracellular Membrane Bound Proteins; U.S. Provisional PatentApplication No. 61/668,157, filed Jul. 5, 2012, entitled ModifiedPolynucleotides for the Production of Intracellular Membrane BoundProteins; U.S. Provisional Patent Application No. 61/681,661, filed Aug.10, 2012, entitled Modified Polynucleotides for the Production ofIntracellular Membrane Bound Proteins; U.S. Provisional PatentApplication No. 61/737,160, filed Dec. 14, 2012, entitled ModifiedPolynucleotides for the Production of Intracellular Membrane BoundProteins; U.S. Provisional Patent Application No. 61/618,911, filed Apr.2, 2012, entitled Modified Polynucleotides for the Production of NuclearProteins; U.S. Provisional Patent Application No. 61/681,667, filed Aug.10, 2012, entitled Modified Polynucleotides for the Production ofNuclear Proteins; U.S. Provisional Patent Application No. 61/737,168,filed Dec. 14, 2012, entitled Modified Polynucleotides for theProduction of Nuclear Proteins; U.S. Provisional Patent Application No.61/618,922, filed Apr. 2, 2012, entitled Modified Polynucleotides forthe Production of Proteins; U.S. Provisional Patent Application No.61/681,675, filed Aug. 10, 2012, entitled Modified Polynucleotides forthe Production of Proteins; U.S. Provisional Patent Application No.61/737,174, filed Dec. 14, 2012, entitled Modified Polynucleotides forthe Production of Proteins; U.S. Provisional Patent Application No.61/618,935, filed Apr. 2, 2012, entitled Modified Polynucleotides forthe Production of Proteins Associated with Human Disease; U.S.Provisional Patent Application No. 61/681,687, filed Aug. 10, 2012,entitled Modified Polynucleotides for the Production of ProteinsAssociated with Human Disease; U.S. Provisional Patent Application No.61/737,184, filed Dec. 14, 2012, entitled Modified Polynucleotides forthe Production of Proteins Associated with Human Disease; U.S.Provisional Patent Application No. 61/618,945, filed Apr. 2, 2012,entitled Modified Polynucleotides for the Production of ProteinsAssociated with Human Disease; U.S. Provisional Patent Application No.61/681,696, filed Aug. 10, 2012, entitled Modified Polynucleotides forthe Production of Proteins Associated with Human Disease; U.S.Provisional Patent Application No. 61/737,191, filed Dec. 14, 2012,entitled Modified Polynucleotides for the Production of ProteinsAssociated with Human Disease; U.S. Provisional Patent Application No.61/618,953, filed Apr. 2, 2012, entitled Modified Polynucleotides forthe Production of Proteins Associated with Human Disease; U.S.Provisional Patent Application No. 61/681,704, filed Aug. 10, 2012,entitled Modified Polynucleotides for the Production of ProteinsAssociated with Human Disease; U.S. Provisional Patent Application No.61/737,203, filed Dec. 14, 2012, entitled Modified Polynucleotides forthe Production of Proteins Associated with Human Disease, InternationalApplication No PCT/US2013/030062, filed Mar. 9, 2013, entitled ModifiedPolynucleotides for the Production of Biologics and Proteins Associatedwith Human Disease; International Application No PCT/US2013/030063,filed Mar. 9, 2013, entitled Modified Polynucloetides; InternationalApplication No. PCT/US2013/030064, entitled Modified Polynucleotides forthe Production of Secreted Proteins; International Application NoPCT/US2013/030059, filed Mar. 9, 2013, entitled Modified Polynucleotidesfor the Production of Membrane Proteins; International Application No.PCT/US2013/030066, filed Mar. 9, 2013, entitled Modified Polynucleotidesfor the Production of Cytoplasmic and Cytoskeletal Proteins;International Application No. PCT/US2013/030067, filed Mar. 9, 2013,entitled Modified Polynucleotides for the Production of NuclearProteins; International Application No. PCT/US2013/030060, filed Mar. 9,2013, entitled Modified Polynucleotides for the Production of Proteins;International Application No. PCT/US2013/030061, filed Mar. 9, 2013,entitled Modified Polynucleotides for the Production of ProteinsAssociated with Human Disease; International Application No.PCT/US2013/030068, filed Mar. 9, 2013, entitled Modified Polynucleotidesfor the Production of Cosmetic Proteins and Peptides; InternationalApplication No. PCT/US2013/030070, filed Mar. 9, 2013, entitled ModifiedPolynucleotides for the Production of Oncology-Related Proteins andPeptides; International Application No. PCT/US2013/031821, filed Mar.15, 2013, entitled In Vivo Production of Proteins; the contents of eachof which are herein incorporated by reference in its entirety.

Protein Cleavage Signals and Sites

In one embodiment, the polypeptides of the present invention may includeat least one protein cleavage signal containing at least one proteincleavage site. The protein cleavage site may be located at theN-terminus, the C-terminus, at any space between the N- and theC-termini such as, but not limited to, half-way between the N- andC-termini, between the N-terminus and the half way point, between thehalf way point and the C-terminus, and combinations thereof.

The polypeptides of the present invention may include, but is notlimited to, a proprotein convertase (or prohormone convertase), thrombinor Factor Xa protein cleavage signal. Proprotein convertases are afamily of nine proteinases, comprising seven basic amino acid-specificsubtilisin-like serine proteinases related to yeast kexin, known asprohormone convertase 1/3 (PC1/3), PC2, furin, PC4, PC5/6, paired basicamino-acid cleaving enzyme 4 (PACE4) and PC7, and two other subtilasesthat cleave at non-basic residues, called subtilisin kexin isozyme 1(SKI-1) and proproteinconvertase subtilisin kexin 9 (PCSK9).

In one embodiment, the primary constructs and mmRNA of the presentinvention may be engineered such that the primary construct or mmRNAcontains at least one encoded protein cleavage signal. The encodedprotein cleavage signal may be located before the start codon, after thestart codon, before the coding region, within the coding region such as,but not limited to, half way in the coding region, between the startcodon and the half way point, between the half way point and the stopcodon, after the coding region, before the stop codon, between two stopcodons, after the stop codon and combinations thereof.

In one embodiment, the primary constructs or mmRNA of the presentinvention may include at least one encoded protein cleavage signalcontaining at least one protein cleavage site. The encoded proteincleavage signal may include, but is not limited to, a proproteinconvertase (or prohormone convertase), thrombin and/or Factor Xa proteincleavage signal. One of skill in the art may use Table 1 above or otherknown methods to determine the appropriate encoded protein cleavagesignal to include in the primary constructs or mmRNA of the presentinvention. For example, starting with a signal sequence and consideringthe codons of Table 1 one can design a signal for the primary constructwhich can produce a protein signal in the resulting polypeptide.

In one embodiment, the polypeptides of the present invention include atleast one protein cleavage signal and/or site.

As a non-limiting example, U.S. Pat. No. 7,374,930 and U.S. Pub. No.20090227660, herein incorporated by reference in their entireties, use afurin cleavage site to cleave the N-terminal methionine of GLP-1 in theexpression product from the Golgi apparatus of the cells. In oneembodiment, the polypeptides of the present invention include at leastone protein cleavage signal and/or site with the proviso that thepolypeptide is not GLP-1.

In one embodiment, the primary constructs or mmRNA of the presentinvention includes at least one encoded protein cleavage signal and/orsite.

In one embodiment, the primary constructs or mmRNA of the presentinvention includes at least one encoded protein cleavage signal and/orsite with the proviso that the primary construct or mmRNA does notencode GLP-1.

In one embodiment, the primary constructs or mmRNA of the presentinvention may include more than one coding region. Where multiple codingregions are present in the primary construct or mmRNA of the presentinvention, the multiple coding regions may be separated by encodedprotein cleavage sites. As a non-limiting example, the primary constructor mmRNA may be signed in an ordered pattern. On such pattern followsAXBY form where A and B are coding regions which may be the same ordifferent coding regions and/or may encode the same or differentpolypeptides, and X and Y are encoded protein cleavage signals which mayencode the same or different protein cleavage signals. A second suchpattern follows the form AXYBZ where A and B are coding regions whichmay be the same or different coding regions and/or may encode the sameor different polypeptides, and X, Y and Z are encoded protein cleavagesignals which may encode the same or different protein cleavage signals.A third pattern follows the form ABXCY where A, B and C are codingregions which may be the same or different coding regions and/or mayencode the same or different polypeptides, and X and Y are encodedprotein cleavage signals which may encode the same or different proteincleavage signals.

In one embodiment, the polypeptides, primary constructs and mmRNA canalso contain sequences that encode protein cleavage sites so that thepolypeptides, primary constructs and mmRNA can be released from acarrier region or a fusion partner by treatment with a specific proteasefor said protein cleavage site.

III. Modifications

Herein, in a polynucleotide (such as a primary construct or an mRNAmolecule), the terms “modification” or, as appropriate, “modified” referto modification with respect to A, G, U or C ribonucleotides. Generally,herein, these terms are not intended to refer to the ribonucleotidemodifications in naturally occurring 5′-terminal mRNA cap moieties. In apolypeptide, the term “modification” refers to a modification ascompared to the canonical set of 20 amino acids, moiety)

The modifications may be various distinct modifications. In someembodiments, the coding region, the flanking regions and/or the terminalregions may contain one, two, or more (optionally different) nucleosideor nucleotide modifications. In some embodiments, a modifiedpolynucleotide, primary construct, or mmRNA introduced to a cell mayexhibit reduced degradation in the cell, as compared to an unmodifiedpolynucleotide, primary construct, or mmRNA.

The polynucleotides, primary constructs, and mmRNA can include anyuseful modification, such as to the sugar, the nucleobase, or theinternucleoside linkage (e.g. to a linking phosphate/to a phosphodiesterlinkage/to the phosphodiester backbone). One or more atoms of apyrimidine nucleobase may be replaced or substituted with optionallysubstituted amino, optionally substituted thiol, optionally substitutedalkyl (e.g., methyl or ethyl), or halo (e.g., chloro or fluoro). Incertain embodiments, modifications (e.g., one or more modifications) arepresent in each of the sugar and the internucleoside linkage.Modifications according to the present invention may be modifications ofribonucleic acids (RNAs) to deoxyribonucleic acids (DNAs), threosenucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids(PNAs), locked nucleic acids (LNAs) or hybrids thereof). Additionalmodifications are described herein.

As described herein, the polynucleotides, primary constructs, and mmRNAof the invention do not substantially induce an innate immune responseof a cell into which the mRNA is introduced. Features of an inducedinnate immune response include 1) increased expression ofpro-inflammatory cytokines, 2) activation of intracellular PRRs (RIG-I,MDA5, etc, and/or 3) termination or reduction in protein translation.

In certain embodiments, it may desirable to intracellularly degrade amodified nucleic acid molecule introduced into the cell. For example,degradation of a modified nucleic acid molecule may be preferable ifprecise timing of protein production is desired. Thus, in someembodiments, the invention provides a modified nucleic acid moleculecontaining a degradation domain, which is capable of being acted on in adirected manner within a cell.

The polynucleotides, primary constructs, and mmRNA can optionallyinclude other agents (e.g., RNAi-inducing agents, RNAi agents, siRNAs,shRNAs, miRNAs, antisense RNAs, ribozymes, catalytic DNA, tRNA, RNAsthat induce triple helix formation, aptamers, vectors, etc.). In someembodiments, the polynucleotides, primary constructs, or mmRNA mayinclude one or more messenger RNAs (mRNAs) and one or more modifiednucleoside or nucleotides (e.g., mmRNA molecules). Details for thesepolynucleotides, primary constructs, and mmRNA follow.

Polynucleotides and Primary Constructs

The polynucleotides, primary constructs, and mmRNA of the inventionincludes a first region of linked nucleosides encoding a polypeptide ofinterest, a first flanking region located at the 5′ terminus of thefirst region, and a second flanking region located at the 3′ terminus ofthe first region.

In some embodiments, the polynucleotide, primary construct, or mmRNA(e.g., the first region, first flanking region, or second flankingregion) includes n number of linked nucleosides having any base, sugar,backbone, building block or other structure or formula, including butnot limited to those of Formulas I through IX or any substructuresthereof as described in International Application PCT/US12/58519 filedOct. 3, 2012 (Attorney Docket Number: M009.20), the contents of whichare incorporated herein by reference in their entirety. Such structuresinclude modifications to the sugar, nucleobase, internucleoside linkage,or combinations thereof.

Combinations of chemical modifications include those taught in includingbut not limited to those described in International ApplicationPCT/US12/58519 filed Oct. 3, 2012 (Attorney Docket Number: M009.20), thecontents of which are incorporated herein by reference in theirentirety.

The synthesis of polynucleotides, primary constructs or mmRNA of thepresent invention may be according to the methods described inInternational Application PCT/US12/58519 filed Oct. 3, 2012 (AttorneyDocket Number: M009.20), the contents of which are incorporated hereinby reference in their entirety.

In some embodiments, the nucleobase selected from the group consistingof cytosine, guanine, adenine, and uracil.

In some embodiments, the modified nucleobase is a modified uracil.Exemplary nucleobases and nucleosides having a modified uracil includepseudouridine (ψ), pyridin-4-one ribonucleoside, 5-aza-uridine,6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s²U),4-thio-uridine (s⁴U), 4-thio-pseudouridine, 2-thio-pseudouridine,5-hydroxy-uridine (ho⁵U), 5-aminoallyl-uridine, 5-halo-uridine (e.g.,5-iodo-uridine or 5-bromo-uridine), 3-methyl-uridine (m³U),5-methoxy-uridine (mo⁵U), uridine 5-oxyacetic acid (cmo⁵U), uridine5-oxyacetic acid methyl ester (mcmo⁵U), 5-carboxymethyl-uridine (cm⁵U),1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm⁵U),5-carboxyhydroxymethyl-uridine methyl ester (mchm⁵U),5-methoxycarbonylmethyl-uridine (mcm⁵U),5-methoxycarbonylmethyl-2-thio-uridine (mcm⁵s²U),5-aminomethyl-2-thio-uridine (nm⁵s²U), 5-methylaminomethyl-uridine(mnm⁵U), 5-methylaminomethyl-2-thio-uridine (mnm⁵s²U),5-methylaminomethyl-2-seleno-uridine (mnm⁵se²U),5-carbamoylmethyl-uridine (ncm⁵U), 5-carboxymethylaminomethyl-uridine(cmnm⁵U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm⁵s²U),5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine(τm⁵U), 1-taurinomethyl-pseudouridine,5-taurinomethyl-2-thio-uridine(τm⁵s²U),1-taurinomethyl-4-thio-pseudouridine, 5-methyl-uridine (m⁵U, i.e.,having the nucleobase deoxythymine), 1-methyl-pseudouridine5-methyl-2-thio-uridine (m⁵s²U), 1-methyl-4-thio-pseudouridine (m¹s⁴ψ),4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m³ψ),2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine,2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine (D),dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m⁵D),2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine,2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine,4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine,3-(3-amino-3-carboxypropyl)uridine (acp³U),1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp³ ψ),5-(isopentenylaminomethyl)uridine (inm⁵U),5-(isopentenylaminomethyl)-2-thio-uridine (inm⁵s²U), α-thio-uridine,2′-O-methyl-uridine (Um), 5,2′-O-dimethyl-uridine (m⁵Um),2′-O-methyl-pseudouridine (ψm), 2-thio-2′-O-methyl-uridine (s²Um),5-methoxycarbonylmethyl-2′-O-methyl-uridine (mcm⁵Um),5-carbamoylmethyl-2′-O-methyl-uridine (ncm⁵Um),5-carboxymethylaminomethyl-2′-O-methyl-uridine (cmnm⁵Um),3,2′-O-dimethyl-uridine (m³Um),5-(isopentenylaminomethyl)-2′-O-methyl-uridine (inm⁵Um), 1-thio-uridine,deoxythymidine, 2′-F-ara-uridine, 2′-F-uridine, 2′-OH-ara-uridine,5-(2-carbomethoxyvinyl) uridine, and 5-[3-(1-E-propenylamino)uridine.

In some embodiments, the modified nucleobase is a modified cytosine.Exemplary nucleobases and nucleosides having a modified cytosine include5-aza-cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine(m³C), N4-acetyl-cytidine (ac⁴C), 5-formyl-cytidine (f⁵C),N4-methyl-cytidine (m⁴C), 5-methyl-cytidine (m⁵C), 5-halo-cytidine(e.g., 5-iodo-cytidine), 5-hydroxymethyl-cytidine (hm⁵C),1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine,2-thio-cytidine (s²C), 2-thio-5-methyl-cytidine,4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine,4-thio-1-methyl-1-deaza-pseudoisocytidine,1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine,5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine,2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine,4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine,lysidine (k₂C), α-thio-cytidine, 2′-O-methyl-cytidine (Cm),5,2′-O-dimethyl-cytidine (m⁵Cm), N4-acetyl-2′-O-methyl-cytidine (ac⁴Cm),N4,2′-O-dimethyl-cytidine (m⁴Cm), 5-formyl-2′-O-methyl-cytidine (f⁵Cm),N4,N4,2′-O-trimethyl-cytidine (m⁴ ₂Cm), 1-thio-cytidine,2′-F-ara-cytidine, 2′-F-cytidine, and 2′-OH-ara-cytidine.

In some embodiments, the modified nucleobase is a modified adenine.Exemplary nucleobases and nucleosides having a modified adenine include2-amino-purine, 2, 6-diaminopurine, 2-amino-6-halo-purine (e.g.,2-amino-6-chloro-purine), 6-halo-purine (e.g., 6-chloro-purine),2-amino-6-methyl-purine, 8-azido-adenosine, 7-deaza-adenine,7-deaza-8-aza-adenine, 7-deaza-2-amino-purine,7-deaza-8-aza-2-amino-purine, 7-deaza-2,6-diaminopurine,7-deaza-8-aza-2,6-diaminopurine, 1-methyl-adenosine (m′A),2-methyl-adenine (m²A), N6-methyl-adenosine (m⁶A),2-methylthio-N6-methyl-adenosine (ms² m⁶A), N6-isopentenyl-adenosine(i⁶A), 2-methylthio-N6-isopentenyl-adenosine (ms²i⁶A),N6-(cis-hydroxyisopentenyl)adenosine (io⁶A),2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine (ms²io⁶A),N6-glycinylcarbamoyl-adenosine (g⁶A), N6-threonylcarbamoyl-adenosine(t⁶A),N6-methyl-N6-threonylcarbamoyl-adenosine2-methylthio-N6-threonylcarbamoyl-(m⁶t⁶A),adenosine (ms²g⁶A), N6,N6-dimethyl-adenosine (m⁶ ₂A),N6-hydroxynorvalylcarbamoyl-adenosine (hn⁶A),2-methylthio-N6-hydroxynorvalylcarbamoyl-adenosine (ms²hn⁶A),N6-acetyl-adenosine (ac⁶A), 7-methyl-adenine, 2-methylthio-adenine,2-methoxy-adenine, α-thio-adenosine, 2′-O-methyl-adenosine (Am),N6,2′-O-dimethyl-adenosine (m⁶Am), N6,N6,2′-O-trimethyl-adenosine (m⁶₂Am), 1,2′-O-dimethyl-adenosine (m¹Am), 2′-O-ribosyladenosine(phosphate) (Ar(p)), 2-amino-N6-methyl-purine, 1-thio-adenosine,8-azido-adenosine, 2′-F-ara-adenosine, 2′-F-adenosine,2′-0H-ara-adenosine, and N6-(19-amino-pentaoxanonadecyl)-adenosine.

In some embodiments, the modified nucleobase is a modified guanine.Exemplary nucleobases and nucleosides having a modified guanine includeinosine (I), 1-methyl-inosine (m¹I), wyosine (imG), methylwyosine(mimG), 4-demethyl-wyosine (imG-14), isowyosine (imG2), wybutosine (yW),peroxywybutosine (o₂yW), hydroxywybutosine (OHyW), undermodifiedhydroxywybutosine (OHyW*), 7-deaza-guanosine, queuosine (Q),epoxyqueuosine (oQ), galactosyl-queuosine (galQ), mannosyl-queuosine(manQ), 7-cyano-7-deaza-guanosine (preQo),7-aminomethyl-7-deaza-guanosine (preQi), archaeosine (G⁺),7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine,6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine (m⁷G),6-thio-7-methyl-guanosine, 7-methyl-inosine, 6-methoxy-guanosine,1-methyl-guanosine (m′G), N2-methyl-guanosine (m²G),N2,N2-dimethyl-guanosine (m² ₂G), N2,7-dimethyl-guanosine (m²′⁷G), N2,N2,7-dimethyl-guanosine 8-oxo-guanosine, 7-methyl-8-oxo-guanosine,1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine,N2,N2-dimethyl-6-thio-guanosine, α-thio-guanosine, 2′-O-methyl-guanosine(Gm), N2-methyl-2′-O-methyl-guanosine (m²Gm),N2,N2-dimethyl-2′-O-methyl-guanosine (m² ₂Gm),1-methyl-2′-O-methyl-guanosine (m′Gm),N2,7-dimethyl-2′-O-methyl-guanosine (m²′⁷Gm), 2′-O-methyl-inosine (Im),1,2′-O-dimethyl-inosine (m¹Im), and 2′-O-ribosylguanosine (phosphate)(Gr(p)).

The nucleobase of the nucleotide can be independently selected from apurine, a pyrimidine, a purine or pyrimidine analog. For example, thenucleobase can each be independently selected from adenine, cytosine,guanine, uracil, or hypoxanthine. In another embodiment, the nucleobasecan also include, for example, naturally-occurring and syntheticderivatives of a base, including pyrazolo[3,4-d]pyrimidines,5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine,hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives ofadenine and guanine, 2-propyl and other alkyl derivatives of adenine andguanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-propynyluracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil(pseudouracil), 4-thiouracil, 8-halo (e.g., 8-bromo), 8-amino, 8-thiol,8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines,5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituteduracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanineand 8-azaadenine, deazaguanine, 7-deazaguanine, 3-deazaguanine,deazaadenine, 7-deazaadenine, 3-deazaadenine, pyrazolo[3,4-d]pyrimidine,imidazo[1,5-a]1,3,5 triazinones, 9-deazapurines,imidazo[4,5-d]pyrazines, thiazolo[4,5-d]pyrimidines, pyrazin-2-ones,1,2,4-triazine, pyridazine; and 1,3,5 triazine. When the nucleotides aredepicted using the shorthand A, G, C, T or U, each letter refers to therepresentative base and/or derivatives thereof, e.g., A includes adenineor adenine analogs, e.g., 7-deaza adenine).

Modified nucleosides and nucleotides (e.g., building block molecules)can be prepared according to the synthetic methods described in Ogata etal., J. Org. Chem. 74:2585-2588 (2009); Purmal et al., Nucl. Acids Res.22(1): 72-78, (1994); Fukuhara et al., Biochemistry, 1(4): 563-568(1962); and Xu et al., Tetrahedron, 48(9): 1729-1740 (1992), each ofwhich are incorporated by reference in their entirety.

The polypeptides, primary constructs, and mmRNA of the invention may ormay not be uniformly modified along the entire length of the molecule.For example, one or more or all types of nucleotide (e.g., purine orpyrimidine, or any one or more or all of A, G, U, C) may or may not beuniformly modified in a polynucleotide of the invention, or in a givenpredetermined sequence region thereof (e.g. one or more of the sequenceregions represented in FIG. 1). In some embodiments, all nucleotides Xin a polynucleotide of the invention (or in a given sequence regionthereof) are modified, wherein X may any one of nucleotides A, G, U, C,or any one of the combinations A+G, A+U, A+C, G+U, G+C, U+C, A+G+U,A+G+C, G+U+C or A+G+C.

Different sugar modifications, nucleotide modifications, and/orinternucleoside linkages (e.g., backbone structures) may exist atvarious positions in the polynucleotide, primary construct, or mmRNA.One of ordinary skill in the art will appreciate that the nucleotideanalogs or other modification(s) may be located at any position(s) of apolynucleotide, primary construct, or mmRNA such that the function ofthe polynucleotide, primary construct, or mmRNA is not substantiallydecreased. A modification may also be a 5′ or 3′ terminal modification.The polynucleotide, primary construct, or mmRNA may contain from about1% to about 100% modified nucleotides (either in relation to overallnucleotide content, or in relation to one or more types of nucleotide,i.e. any one or more of A, G, U or C) or any intervening percentage(e.g., from 1% to 20%, from 1% to 25%, from 1% to 50%, from 1% to 60%,from 1% to 70%, from 1% to 80%, from 1% to 90%, from 1% to 95%, from 10%to 20%, from 10% to 25%, from 10% to 50%, from 10% to 60%, from 10% to70%, from 10% to 80%, from 10% to 90%, from 10% to 95%, from 10% to100%, from 20% to 25%, from 20% to 50%, from 20% to 60%, from 20% to70%, from 20% to 80%, from 20% to 90%, from 20% to 95%, from 20% to100%, from 50% to 60%, from 50% to 70%, from 50% to 80%, from 50% to90%, from 50% to 95%, from 50% to 100%, from 70% to 80%, from 70% to90%, from 70% to 95%, from 70% to 100%, from 80% to 90%, from 80% to95%, from 80% to 100%, from 90% to 95%, from 90% to 100%, and from 95%to 100%).

In some embodiments, the polynucleotide, primary construct, or mmRNAincludes a modified pyrimidine (e.g., a modified uracil/uridine/U ormodified cytosine/cytidine/C). In some embodiments, the uracil oruridine (generally: U) in the polynucleotide, primary construct, ormmRNA molecule may be replaced with from about 1% to about 100% of amodified uracil or modified uridine (e.g., from 1% to 20%, from 1% to25%, from 1% to 50%, from 1% to 60%, from 1% to 70%, from 1% to 80%,from 1% to 90%, from 1% to 95%, from 10% to 20%, from 10% to 25%, from10% to 50%, from 10% to 60%, from 10% to 70%, from 10% to 80%, from 10%to 90%, from 10% to 95%, from 10% to 100%, from 20% to 25%, from 20% to50%, from 20% to 60%, from 20% to 70%, from 20% to 80%, from 20% to 90%,from 20% to 95%, from 20% to 100%, from 50% to 60%, from 50% to 70%,from 50% to 80%, from 50% to 90%, from 50% to 95%, from 50% to 100%,from 70% to 80%, from 70% to 90%, from 70% to 95%, from 70% to 100%,from 80% to 90%, from 80% to 95%, from 80% to 100%, from 90% to 95%,from 90% to 100%, and from 95% to 100% of a modified uracil or modifieduridine). The modified uracil or uridine can be replaced by a compoundhaving a single unique structure or by a plurality of compounds havingdifferent structures (e.g., 2, 3, 4 or more unique structures, asdescribed herein).

In some embodiments, the cytosine or cytidine (generally: C) in thepolynucleotide, primary construct, or mmRNA molecule may be replacedwith from about 1% to about 100% of a modified cytosine or modifiedcytidine (e.g., from 1% to 20%, from 1% to 25%, from 1% to 50%, from 1%to 60%, from 1% to 70%, from 1% to 80%, from 1% to 90%, from 1% to 95%,from 10% to 20%, from 10% to 25%, from 10% to 50%, from 10% to 60%, from10% to 70%, from 10% to 80%, from 10% to 90%, from 10% to 95%, from 10%to 100%, from 20% to 25%, from 20% to 50%, from 20% to 60%, from 20% to70%, from 20% to 80%, from 20% to 90%, from 20% to 95%, from 20% to100%, from 50% to 60%, from 50% to 70%, from 50% to 80%, from 50% to90%, from 50% to 95%, from 50% to 100%, from 70% to 80%, from 70% to90%, from 70% to 95%, from 70% to 100%, from 80% to 90%, from 80% to95%, from 80% to 100%, from 90% to 95%, from 90% to 100%, and from 95%to 100% of a modified cytosine or modified cytidine). The modifiedcytosine or cytidine can be replaced by a compound having a singleunique structure or by a plurality of compounds having differentstructures (e.g., 2, 3, 4 or more unique structures, as describedherein).

In some embodiments, the polynucleotide, primary construct, or mmRNA istranslatable.

Other components of polynucleotides, primary constructs, and mmRNA areoptional, and are beneficial in some embodiments. For example, a 5′untranslated region (UTR) and/or a 3′UTR are provided, wherein either orboth may independently contain one or more different nucleotidemodifications. In such embodiments, nucleotide modifications may also bepresent in the translatable region. Also provided are polynucleotides,primary constructs, and mmRNA containing a Kozak sequence.

In some embodiments, at least 25% of the cytosines are replaced by acompound of Formula (b10)-(b14) (e.g., at least about 30%, at leastabout 35%, at least about 40%, at least about 45%, at least about 50%,at least about 55%, at least about 60%, at least about 65%, at leastabout 70%, at least about 75%, at least about 80%, at least about 85%,at least about 90%, at least about 95%, or about 100%).

In some embodiments, at least 25% of the uracils are replaced by acompound of Formula (b1)-(b9) (e.g., at least about 30%, at least about35%, at least about 40%, at least about 45%, at least about 50%, atleast about 55%, at least about 60%, at least about 65%, at least about70%, at least about 75%, at least about 80%, at least about 85%, atleast about 90%, at least about 95%, or about 100%).

In some embodiments, at least 25% of the cytosines are replaced by acompound of Formula (b10)-(b14), and at least 25% of the uracils arereplaced by a compound of Formula (b1)-(b9) (e.g., at least about 30%,at least about 35%, at least about 40%, at least about 45%, at leastabout 50%, at least about 55%, at least about 60%, at least about 65%,at least about 70%, at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 95%, or about 100%).

IV. Pharmaceutical Compositions Formulation, Administration, Deliveryand Dosing

The present invention provides polynucleotides, primary constructs andmmRNA compositions and complexes in combination with one or morepharmaceutically acceptable excipients. Pharmaceutical compositions mayoptionally comprise one or more additional active substances, e.g.therapeutically and/or prophylactically active substances. Generalconsiderations in the formulation and/or manufacture of pharmaceuticalagents may be found, for example, in Remington: The Science and Practiceof Pharmacy 21^(st) ed., Lippincott Williams & Wilkins, 2005(incorporated herein by reference in its entirety).

In some embodiments, compositions are administered to humans, humanpatients or subjects. For the purposes of the present disclosure, thephrase “active ingredient” generally refers to polynucleotides, primaryconstructs and mmRNA to be delivered as described herein.

Although the descriptions of pharmaceutical compositions provided hereinare principally directed to pharmaceutical compositions which aresuitable for administration to humans, it will be understood by theskilled artisan that such compositions are generally suitable foradministration to any other animal, e.g., to non-human animals, e.g.non-human mammals. Modification of pharmaceutical compositions suitablefor administration to humans in order to render the compositionssuitable for administration to various animals is well understood, andthe ordinarily skilled veterinary pharmacologist can design and/orperform such modification with merely ordinary, if any, experimentation.Subjects to which administration of the pharmaceutical compositions iscontemplated include, but are not limited to, humans and/or otherprimates; mammals, including commercially relevant mammals such ascattle, pigs, horses, sheep, cats, dogs, mice, and/or rats; and/orbirds, including commercially relevant birds such as poultry, chickens,ducks, geese, and/or turkeys.

Formulations of the pharmaceutical compositions described herein may beprepared by any method known or hereafter developed in the art ofpharmacology. In general, such preparatory methods include the step ofbringing the active ingredient into association with an excipient and/orone or more other accessory ingredients, and then, if necessary and/ordesirable, dividing, shaping and/or packaging the product into a desiredsingle- or multi-dose unit.

A pharmaceutical composition in accordance with the invention may beprepared, packaged, and/or sold in bulk, as a single unit dose, and/oras a plurality of single unit doses. As used herein, a “unit dose” isdiscrete amount of the pharmaceutical composition comprising apredetermined amount of the active ingredient. The amount of the activeingredient is generally equal to the dosage of the active ingredientwhich would be administered to a subject and/or a convenient fraction ofsuch a dosage such as, for example, one-half or one-third of such adosage.

Relative amounts of the active ingredient, the pharmaceuticallyacceptable excipient, and/or any additional ingredients in apharmaceutical composition in accordance with the invention will vary,depending upon the identity, size, and/or condition of the subjecttreated and further depending upon the route by which the composition isto be administered. By way of example, the composition may comprisebetween 0.1% and 100%, e.g., between 0.5 and 50%, between 1-30%, between5-80%, at least 80% (w/w) active ingredient.

Any of the polynucleotides, primary constructs and mmRNA describedherein may be formulated as described in International Application NoPCT/US2012/069610, filed Dec. 14, 2012, entitled Modified Nucleoside,Nucleotide, and Nucleic Acid Compositions, the contents of which isherein incorporated by reference in its entirety.

Formulations

The polynucleotide, primary construct, and mmRNA of the invention can beformulated using one or more excipients to: (1) increase stability; (2)increase cell transfection; (3) permit the sustained or delayed release(e.g., from a depot formulation of the polynucleotide, primaryconstruct, or mmRNA); (4) alter the biodistribution (e.g., target thepolynucleotide, primary construct, or mmRNA to specific tissues or celltypes); (5) increase the translation of encoded protein in vivo; and/or(6) alter the release profile of encoded protein in vivo. In addition totraditional excipients such as any and all solvents, dispersion media,diluents, or other liquid vehicles, dispersion or suspension aids,surface active agents, isotonic agents, thickening or emulsifyingagents, preservatives, excipients of the present invention can include,without limitation, lipidoids, liposomes, lipid nanoparticles, polymers,lipoplexes, core-shell nanoparticles, peptides, proteins, cellstransfected with polynucleotide, primary construct, or mmRNA (e.g., fortransplantation into a subject), hyaluronidase, nanoparticle mimics andcombinations thereof. Accordingly, the formulations of the invention caninclude one or more excipients, each in an amount that togetherincreases the stability of the polynucleotide, primary construct, ormmRNA, increases cell transfection by the polynucleotide, primaryconstruct, or mmRNA, increases the expression of polynucleotide, primaryconstruct, or mmRNA encoded protein, and/or alters the release profileof polynucleotide, primary construct, or mmRNA encoded proteins.Further, the primary construct and mmRNA of the present invention may beformulated using self-assembled nucleic acid nanoparticles.

Formulations of the pharmaceutical compositions described herein may beprepared by any method known or hereafter developed in the art ofpharmacology. In general, such preparatory methods include the step ofassociating the active ingredient with an excipient and/or one or moreother accessory ingredients.

A pharmaceutical composition in accordance with the present disclosuremay be prepared, packaged, and/or sold in bulk, as a single unit dose,and/or as a plurality of single unit doses. As used herein, a “unitdose” refers to a discrete amount of the pharmaceutical compositioncomprising a predetermined amount of the active ingredient. The amountof the active ingredient may generally be equal to the dosage of theactive ingredient which would be administered to a subject and/or aconvenient fraction of such a dosage including, but not limited to,one-half or one-third of such a dosage.

Relative amounts of the active ingredient, the pharmaceuticallyacceptable excipient, and/or any additional ingredients in apharmaceutical composition in accordance with the present disclosure mayvary, depending upon the identity, size, and/or condition of the subjectbeing treated and further depending upon the route by which thecomposition is to be administered. For example, the composition maycomprise between 0.1% and 99% (w/w) of the active ingredient.

In some embodiments, the formulations described herein may contain atleast one mmRNA. As a non-limiting example, the formulations may contain1, 2, 3, 4 or 5 mmRNA. In one embodiment the formulation may containmodified mRNA encoding proteins selected from categories such as, butnot limited to, human proteins, veterinary proteins, bacterial proteins,biological proteins, antibodies, immunogenic proteins, therapeuticpeptides and proteins, secreted proteins, plasma membrane proteins,cytoplasmic and cytoskeletal proteins, intrancellular membrane boundproteins, nuclear proteins, proteins associated with human diseaseand/or proteins associated with non-human diseases. In one embodiment,the formulation contains at least three modified mRNA encoding proteins.In one embodiment, the formulation contains at least five modified mRNAencoding proteins.

Pharmaceutical formulations may additionally comprise a pharmaceuticallyacceptable excipient, which, as used herein, includes, but is notlimited to, any and all solvents, dispersion media, diluents, or otherliquid vehicles, dispersion or suspension aids, surface active agents,isotonic agents, thickening or emulsifying agents, preservatives, andthe like, as suited to the particular dosage form desired. Variousexcipients for formulating pharmaceutical compositions and techniquesfor preparing the composition are known in the art (see Remington: TheScience and Practice of Pharmacy, 21^(st) Edition, A. R. Gennaro,Lippincott, Williams & Wilkins, Baltimore, Md., 2006; incorporatedherein by reference). The use of a conventional excipient medium may becontemplated within the scope of the present disclosure, except insofaras any conventional excipient medium may be incompatible with asubstance or its derivatives, such as by producing any undesirablebiological effect or otherwise interacting in a deleterious manner withany other component(s) of the pharmaceutical composition.

In some embodiments, the particle size of the lipid nanoparticle may beincreased and/or decreased. The change in particle size may be able tohelp counter biological reaction such as, but not limited to,inflammation or may increase the biological effect of the modified mRNAdelivered to mammals.

Pharmaceutically acceptable excipients used in the manufacture ofpharmaceutical compositions include, but are not limited to, inertdiluents, surface active agents and/or emulsifiers, preservatives,buffering agents, lubricating agents, and/or oils. Such excipients mayoptionally be included in the pharmaceutical formulations of theinvention.

Lipidoids

The synthesis of lipidoids has been extensively described andformulations containing these compounds are particularly suited fordelivery of polynucleotides, primary constructs or mmRNA (see Mahon etal., Bioconjug Chem. 2010 21:1448-1454; Schroeder et al., J Intern Med.2010 267:9-21; Akinc et al., Nat Biotechnol. 2008 26:561-569; Love etal., Proc Natl Acad Sci USA. 2010 107:1864-1869; Siegwart et al., ProcNatl Acad Sci USA. 2011 108:12996-3001; all of which are incorporatedherein in their entireties).

While these lipidoids have been used to effectively deliver doublestranded small interfering RNA molecules in rodents and non-humanprimates (see Akinc et al., Nat Biotechnol. 2008 26:561-569;Frank-Kamenetsky et al., Proc Natl Acad Sci USA. 2008 105:11915-11920;Akinc et al., Mol Ther. 2009 17:872-879; Love et al., Proc Natl Acad SciUSA. 2010 107:1864-1869; Leuschner et al., Nat Biotechnol. 201129:1005-1010; all of which is incorporated herein in their entirety),the present disclosure describes their formulation and use in deliveringsingle stranded polynucleotides, primary constructs, or mmRNA.Complexes, micelles, liposomes or particles can be prepared containingthese lipidoids and therefore, can result in an effective delivery ofthe polynucleotide, primary construct, or mmRNA, as judged by theproduction of an encoded protein, following the injection of a lipidoidformulation via localized and/or systemic routes of administration.Lipidoid complexes of polynucleotides, primary constructs, or mmRNA canbe administered by various means including, but not limited to,intravenous, intramuscular, or subcutaneous routes.

In vivo delivery of nucleic acids may be affected by many parameters,including, but not limited to, the formulation composition, nature ofparticle PEGylation, degree of loading, oligonucleotide to lipid ratio,and biophysical parameters such as particle size (Akinc et al., MolTher. 2009 17:872-879; herein incorporated by reference in itsentirety). As an example, small changes in the anchor chain length ofpoly(ethylene glycol) (PEG) lipids may result in significant effects onin vivo efficacy. Formulations with the different lipidoids, including,but not limited topenta[3-(1-laurylaminopropionyl)]-triethylenetetramine hydrochloride(TETA-5LAP; aka 98N12-5, see Murugaiah et al., Analytical Biochemistry,401:61 (2010)), C12-200 (including derivatives and variants), and MD1,can be tested for in vivo activity.

The lipidoid referred to herein as “98N12-5” is disclosed by Akinc etal., Mol Ther. 2009 17:872-879 and is incorporated by reference in itsentirety. (See FIG. 2)

The lipidoid referred to herein as “C12-200” is disclosed by Love etal., Proc Natl Acad Sci USA. 2010 107:1864-1869 (see FIG. 2) and Liu andHuang, Molecular Therapy. 2010 669-670 (see FIG. 2); both of which areherein incorporated by reference in their entirety. The lipidoidformulations can include particles comprising either 3 or 4 or morecomponents in addition to polynucleotide, primary construct, or mmRNA.As an example, formulations with certain lipidoids, include, but are notlimited to, 98N12-5 and may contain 42% lipidoid, 48% cholesterol and10% PEG (C14 alkyl chain length). As another example, formulations withcertain lipidoids, include, but are not limited to, C12-200 and maycontain 50% lipidoid, 10% disteroylphosphatidyl choline, 38.5%cholesterol, and 1.5% PEG-DMG.

In one embodiment, a polynucleotide, primary construct, or mmRNAformulated with a lipidoid for systemic intravenous administration cantarget the liver. For example, a final optimized intravenous formulationusing polynucleotide, primary construct, or mmRNA, and comprising alipid molar composition of 42% 98N12-5, 48% cholesterol, and 10%PEG-lipid with a final weight ratio of about 7.5 to 1 total lipid topolynucleotide, primary construct, or mmRNA, and a C14 alkyl chainlength on the PEG lipid, with a mean particle size of roughly 50-60 nm,can result in the distribution of the formulation to be greater than 90%to the liver (see, Akinc et al., Mol Ther. 2009 17:872-879; hereinincorporated in its entirety). In another example, an intravenousformulation using a C12-200 (see U.S. provisional application 61/175,770and published international application WO2010129709, each of which isherein incorporated by reference in their entirety) lipidoid may have amolar ratio of 50/10/38.5/1.5 of C12-200/disteroylphosphatidylcholine/cholesterol/PEG-DMG, with a weight ratio of 7 to 1 total lipidto polynucleotide, primary construct, or mmRNA, and a mean particle sizeof 80 nm may be effective to deliver polynucleotide, primary construct,or mmRNA to hepatocytes (see, Love et al., Proc Natl Acad Sci USA. 2010107:1864-1869 herein incorporated by reference). In another embodiment,an MD1 lipidoid-containing formulation may be used to effectivelydeliver polynucleotide, primary construct, or mmRNA to hepatocytes invivo. The characteristics of optimized lipidoid formulations forintramuscular or subcutaneous routes may vary significantly depending onthe target cell type and the ability of formulations to diffuse throughthe extracellular matrix into the blood stream. While a particle size ofless than 150 nm may be desired for effective hepatocyte delivery due tothe size of the endothelial fenestrae (see, Akinc et al., Mol Ther. 200917:872-879 herein incorporated by reference), use of alipidoid-formulated polynucleotide, primary construct, or mmRNA todeliver the formulation to other cells types including, but not limitedto, endothelial cells, myeloid cells, and muscle cells may not besimilarly size-limited. Use of lipidoid formulations to deliver siRNA invivo to other non-hepatocyte cells such as myeloid cells and endotheliumhas been reported (see Akinc et al., Nat Biotechnol. 2008 26:561-569;Leuschner et al., Nat Biotechnol. 2011 29:1005-1010; Cho et al. Adv.Funct. Mater. 2009 19:3112-3118; 8^(th) International Judah FolkmanConference, Cambridge, Mass. Oct. 8-9, 2010 herein incorporated byreference in its entirety). Effective delivery to myeloid cells, such asmonocytes, lipidoid formulations may have a similar component molarratio. Different ratios of lipidoids and other components including, butnot limited to, disteroylphosphatidyl choline, cholesterol and PEG-DMG,may be used to optimize the formulation of the polynucleotide, primaryconstruct, or mmRNA for delivery to different cell types including, butnot limited to, hepatocytes, myeloid cells, muscle cells, etc. Forexample, the component molar ratio may include, but is not limited to,50% C12-200, 10% disteroylphosphatidyl choline, 38.5% cholesterol, and%1.5 PEG-DMG (see Leuschner et al., Nat Biotechnol 2011 29:1005-1010;herein incorporated by reference in its entirety). The use of lipidoidformulations for the localized delivery of nucleic acids to cells (suchas, but not limited to, adipose cells and muscle cells) via eithersubcutaneous or intramuscular delivery, may not require all of theformulation components desired for systemic delivery, and as such maycomprise only the lipidoid and the polynucleotide, primary construct, ormmRNA.

Combinations of different lipidoids may be used to improve the efficacyof polynucleotide, primary construct, or mmRNA directed proteinproduction as the lipidoids may be able to increase cell transfection bythe polynucleotide, primary construct, or mmRNA; and/or increase thetranslation of encoded protein (see Whitehead et al., Mol. Ther. 2011,19:1688-1694, herein incorporated by reference in its entirety).

Liposomes, Lipoplexes, and Lipid Nanoparticles

The polynucleotide, primary construct, and mmRNA of the invention can beformulated using one or more liposomes, lipoplexes, or lipidnanoparticles. In one embodiment, pharmaceutical compositions ofpolynucleotide, primary construct, or mmRNA include liposomes. Liposomesare artificially-prepared vesicles which may primarily be composed of alipid bilayer and may be used as a delivery vehicle for theadministration of nutrients and pharmaceutical formulations. Liposomescan be of different sizes such as, but not limited to, a multilamellarvesicle (MLV) which may be hundreds of nanometers in diameter and maycontain a series of concentric bilayers separated by narrow aqueouscompartments, a small unicellular vesicle (SUV) which may be smallerthan 50 nm in diameter, and a large unilamellar vesicle (LUV) which maybe between 50 and 500 nm in diameter. Liposome design may include, butis not limited to, opsonins or ligands in order to improve theattachment of liposomes to unhealthy tissue or to activate events suchas, but not limited to, endocytosis. Liposomes may contain a low or ahigh pH in order to improve the delivery of the pharmaceuticalformulations.

The formation of liposomes may depend on the physicochemicalcharacteristics such as, but not limited to, the pharmaceuticalformulation entrapped and the liposomal ingredients, the nature of themedium in which the lipid vesicles are dispersed, the effectiveconcentration of the entrapped substance and its potential toxicity, anyadditional processes involved during the application and/or delivery ofthe vesicles, the optimization size, polydispersity and the shelf-lifeof the vesicles for the intended application, and the batch-to-batchreproducibility and possibility of large-scale production of safe andefficient liposomal products.

In one embodiment, pharmaceutical compositions described herein mayinclude, without limitation, liposomes such as those formed from1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA) liposomes, DiLa2liposomes from Marina Biotech (Bothell, Wash.),1,2-dilinoleyloxy-3-dimethylaminopropane (DLin-DMA),2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA),and MC3 (US20100324120; herein incorporated by reference in itsentirety) and liposomes which may deliver small molecule drugs such as,but not limited to, DOXIL® from Janssen Biotech, Inc. (Horsham, Pa.).

In one embodiment, pharmaceutical compositions described herein mayinclude, without limitation, liposomes such as those formed from thesynthesis of stabilized plasmid-lipid particles (SPLP) or stabilizednucleic acid lipid particle (SNALP) that have been previously describedand shown to be suitable for oligonucleotide delivery in vitro and invivo (see Wheeler et al. Gene Therapy. 1999 6:271-281; Zhang et al. GeneTherapy. 1999 6:1438-1447; Jeffs et al. Pharm Res. 2005 22:362-372;Morrissey et al., Nat Biotechnol. 2005 2:1002-1007; Zimmermann et al.,Nature. 2006 441:111-114; Heyes et al. J Contr Rel. 2005 107:276-287;Semple et al. Nature Biotech. 2010 28:172-176; Judge et al. J ClinInvest. 2009 119:661-673; deFougerolles Hum Gene Ther. 2008 19:125-132;all of which are incorporated herein in their entireties). The originalmanufacture method by Wheeler et al. was a detergent dialysis method,which was later improved by Jeffs et al. and is referred to as thespontaneous vesicle formation method. The liposome formulations arecomposed of 3 to 4 lipid components in addition to the polynucleotide,primary construct, or mmRNA. As an example a liposome can contain, butis not limited to, 55% cholesterol, 20% disteroylphosphatidyl choline(DSPC), 10% PEG-S-DSG, and 15% 1,2-dioleyloxy-N,N-dimethylaminopropane(DODMA), as described by Jeffs et al. As another example, certainliposome formulations may contain, but are not limited to, 48%cholesterol, 20% DSPC, 2% PEG-c-DMA, and 30% cationic lipid, where thecationic lipid can be 1,2-distearloxy-N,N-dimethylaminopropane (DSDMA),DODMA, DLin-DMA, or 1,2-dilinolenyloxy-3-dimethylaminopropane (DLenDMA),as described by Heyes et al.

In one embodiment, the polynucleotides, primary constructs and/or mmRNAmay be formulated in a lipid vesicle which may have crosslinks betweenfunctionalized lipid bilayers.

In one embodiment, the polynucleotides, primary constructs and/or mmRNAmay be formulated in a lipid-polycation complex. The formation of thelipid-polycation complex may be accomplished by methods known in the artand/or as described in U.S. Pub. No. 20120178702, herein incorporated byreference in its entirety. As a non-limiting example, the polycation mayinclude a cationic peptide or a polypeptide such as, but not limited to,polylysine, polyornithine and/or polyarginine. In another embodiment,the polynucleotides, primary constructs and/or mmRNA may be formulatedin a lipid-polycation complex which may further include a neutral lipidsuch as, but not limited to, cholesterol or dioleoylphosphatidylethanolamine (DOPE).

The liposome formulation may be influenced by, but not limited to, theselection of the cationic lipid component, the degree of cationic lipidsaturation, the nature of the PEGylation, ratio of all components andbiophysical parameters such as size. In one example by Semple et al.(Semple et al. Nature Biotech. 2010 28:172-176), the liposomeformulation was composed of 57.1% cationic lipid, 7.1%dipalmitoylphosphatidylcholine, 34.3% cholesterol, and 1.4% PEG-c-DMA.As another example, changing the composition of the cationic lipid couldmore effectively deliver siRNA to various antigen presenting cells(Basha et al. Mol Ther. 2011 19:2186-2200; herein incorporated byreference in its entirety).

In some embodiments, the ratio of PEG in the LNP formulations may beincreased or decreased and/or the carbon chain length of the PEG lipidmay be modified from C14 to C18 to alter the pharmacokinetics and/orbiodistribution of the LNP formulations. As a non-limiting example, LNPformulations may contain 1-5% of the lipid molar ratio of PEG-c-DOMG ascompared to the cationic lipid, DSPC and cholesterol. In anotherembodiment the PEG-c-DOMG may be replaced with a PEG lipid such as, butnot limited to, PEG-DSG (1,2-Distearoyl-sn-glycerol, methoxypolyethyleneglycol) or PEG-DPG (1,2-Dipalmitoyl-sn-glycerol, methoxypolyethyleneglycol). The cationic lipid may be selected from any lipid known in theart such as, but not limited to, DLin-MC3-DMA, DLin-DMA, C12-200 andDLin-KC2-DMA.

In one embodiment, the cationic lipid may be selected from, but notlimited to, a cationic lipid described in International Publication Nos.WO2012040184, WO2011153120, WO2011149733, WO2011090965, WO2011043913,WO2011022460, WO2012061259, WO2012054365, WO2012044638, WO2010080724,WO201021865 and WO2008103276, U.S. Pat. Nos. 7,893,302 and 7,404,969 andUS Patent Publication No. US20100036115; each of which is hereinincorporated by reference in their entirety. In another embodiment, thecationic lipid may be selected from, but not limited to, formula Adescribed in International Publication Nos. WO2012040184, WO2011153120,WO2011149733, WO2011090965, WO2011043913, WO2011022460, WO2012061259,WO2012054365 and WO2012044638; each of which is herein incorporated byreference in their entirety. In yet another embodiment, the cationiclipid may be selected from, but not limited to, formula CLI-CLXXIX ofInternational Publication No. WO2008103276, formula CLI-CLXXIX of U.S.Pat. No. 7,893,302, formula CLI-CLXXXXII of U.S. Pat. No. 7,404,969 andformula I-VI of US Patent Publication No. 0520100036115; each of whichis herein incorporated by reference in their entirety. As a non-limitingexample, the cationic lipid may be selected from(20Z,23Z)—N,N-dimethylnonacosa-20,23-dien-10-amine,(17Z,20Z)—N,N-dimemylhexacosa-17,20-dien-9-amine, (1Z,19Z)—N5N˜dimethylpentacosa˜l 6, 19-dien-8-amine,(13Z,16Z)—N,N-dimethyldocosa-13J16-dien-5-amine,(12Z,15Z)—NJN-dimethylhenicosa-12,15-dien-4-amine,(14Z,17Z)—N,N-dimethyltricosa-14,17-dien-6-amine,(15Z,18Z)—N,N-dimethyltetracosa-15,18-dien-7-amine,(18Z,21Z)—N,N-dimethylheptacosa-18,21-dien-10-amine,(15Z,18Z)—N,N-dimethyltetracosa-15,18-dien-5-amine,(14Z,17Z)—N,N-dimethyltricosa-14,17-dien-4-amine,(19Z,22Z)—N,N-dimeihyloctacosa-19,22-dien-9-amine,(18Z,21Z)—N,N-dimethylheptacosa-18,21-dien-8-amine,(17Z,20Z)—N,N-dimethylhexacosa-17,20-dien-7-amine,(16Z;19Z)—N,N-dimethylpentacosa-16,19-dien-6-amine,(22Z,25Z)—N,N-dimethylhentriaconta-22,25-dien-10-amine,(21Z,24Z)—N;N-dimethyltriaconta-21,24-dien-9-amine,(18Z)—N,N-dimetylheptacos-18-en-10-amine,(17Z)—N,N-dimethylhexacos-17-en-9-amine,(19Z,22Z)—NJN-dimethyloctacosa-19,22-dien-7-amine,N,N-dimethylheptacosan-10-amine,(20Z,23Z)—N-ethyl-N-methylnonacosa-20J23-dien-1 0-amine, 1-[(11Z,14Z)-1-nonylicosa-1 1,14-dien-1-yl] pyrrolidine,(20Z)—N,N-dimethylheptacos-20-en-1 0-amine, (15Z)—N,N-dimethyleptacos-15-en-1 0-amine, (14Z)—N,N-dimethylnonacos-14-en-1 0-amine,(17Z)—N,N-dimethylnonacos-17-en-1 0-amine,(24Z)—N,N-dimethyltritriacont-24-en-1 0-amine,(20Z)—N,N-dimethylnonacos-20-en-1 0-amine,(22Z)—N,N-dimethylhentriacont-22-en-1 0-amine,(16Z)—N,N-dimethylpentacos-16-en-8-amine,(12Z,15Z)—N,N-dimethyl-2-nonylhenico sa-12, 15-dien-1-amine,(13Z,16Z)—N,N-dimethyl-3-nonyldocosa-1 3, 16-dien-1-amine,N,N-dimethyl-1-[(1 S,2R)-2-octylcyclopropyl] eptadecan-8-amine,1-[(1S,2R)-2-hexylcyclopropyl]-N,N-dimethylnonadecan-10-amine,N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]nonadecan-10-amine,N,N-dimethyl-21˜[(1S,2R)-2-octylcyclopropyl]henicosan-1 0-amine,N,N-dimethyl-1-[(1 S,2S)-2-{[(1R,2R)-2-pentylcycIopropyl]methyl}cyclopropyl]nonadecan-10-amine,N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]hexadecan-8-amine,N,N-dimethyH-[(1R,2S)-2-undecyIcyclopropyl]tetradecan-5-amine,N,N-dimethyl-3-{7-[(1S,2R)-2-octylcyclopropyl]heptyl} dodecan-1-amine,1-[(1R,2S)-2-heptylcyclopropy 1]-N,N-dimethyloctadecan-9-amine,1-[(1S,2R)-2-decylcyclopropyl]-N,N-dimethylpentadecan-6-amine,N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]pentadecan-8-amine,R—N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-(octyloxy)propan-2-amine,S—N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-(octyloxy)propan-2-amine,1-{2-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-1-[(octyloxy)methyl]ethyl}pyrrolidine,(2S)—N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-[(5Z)-oct-5-en-1-yloxy]propan-2-amine,1-{2-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-1-[(octyloxy)methyl]ethyl}azetidine,(2S)-1-(hexyloxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine,(2S)-1-(heptyloxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine,N,N-dimethyl-1-(nonyloxy)-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine,N,N-dimethyl-1-[(9Z)-octadec-9-en-1-yloxy]-3-(octyloxy)propan-2-amine(Compound 9);(2S)—N,N-dimethyl-1-[(6Z,9Z,12Z)-octadeca-6,9,12-trien-1-yloxy]-3-(octyloxy)propan-2-amine,(2S)-1-[(11Z,14Z)-icosa-11,14-dien-1-yloxy]-N,N-dimethyl-3-(pentyloxy)propan-2-amine,(2S)-1-(hexyloxy)-3-[(11Z,14Z)-icosa-11,14-dien-1-yloxy]-N,N-dimethylpropan-2-amine,1-[(11Z,14Z)-icosa-11,14-dien-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine,1-[(13Z,16Z)-docosa-13,16-dien-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine,(2S)-1-[(13Z,16Z)-docosa-13,16-dien-1-yloxy]-3-(hexyloxy)-N,N-dimethylpropan-2-amine,(2S)-1-[(13Z)-docos-13-en-1-yloxy]-3-(hexyloxy)-N,N-dimethylpropan-2-amine,1-[(13Z)-docos-13-en-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine,1-[(9Z)-hexadec-9-en-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine,(2R)—N,N-dimethyl-H(1-metoyloctyl)oxy]-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine,(2R)-1-[(3,7-dimethyloctyl)oxy]-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine,N,N-dimethyl-1-(octyloxy)-3-({8-[(1S,2S)-2-{[(1R,2R)-2-pentylcyclopropyl]methyl}cyclopropyl]octyl}oxy)propan-2-amine,N,N-dimethyl-1-{[8-(2-oc1ylcyclopropyl)octyl]oxy}-3-(octyloxy)propan-2-amineand (11E,20Z,23Z)—N;N-dimethylnonacosa-11,20,2-trien-10-amine or apharmaceutically acceptable salt or stereoisomer thereof.

In one embodiment, the cationic lipid may be synthesized by methodsknown in the art and/or as described in International Publication Nos.WO2012040184, WO2011153120, WO2011149733, WO2011090965, WO2011043913,WO2011022460, WO2012061259, WO2012054365, WO2012044638, WO2010080724 andWO201021865; each of which is herein incorporated by reference in theirentirety.

In one embodiment, the LNP formulations of the polynucleotides, primaryconstructs and/or mmRNA may contain PEG-c-DOMG 3% lipid molar ratio. Inanother embodiment, the LNP formulations of the polynucleotides, primaryconstructs and/or mmRNA may contain PEG-c-DOMG 1.5% lipid molar ratio.

In one embodiment, the pharmaceutical compositions of thepolynucleotides, primary constructs and/or mmRNA may include at leastone of the PEGylated lipids described in International Publication No.2012099755, herein incorporated by reference.

In one embodiment, the LNP formulation may contain PEG-DMG 2000(1,2-dimyristoyl-sn-glycero-3-phophoethanolamine-N-[methoxy(polyethyleneglycol)-2000). In one embodiment, the LNP formulation may containPEG-DMG 2000, a cationic lipid known in the art and at least one othercomponent. In another embodiment, the LNP formulation may containPEG-DMG 2000, a cationic lipid known in the art, DSPC and cholesterol.As a non-limiting example, the LNP formulation may contain PEG-DMG 2000,DLin-DMA, DSPC and cholesterol. As another non-limiting example the LNPformulation may contain PEG-DMG 2000, DLin-DMA, DSPC and cholesterol ina molar ratio of 2:40:10:48 (see Geall et al., Nonviral delivery ofself-amplifying RNA vaccines, PNAS 2012; PMID: 22908294).

In one embodiment, the LNP formulation may be formulated by the methodsdescribed in International Publication Nos. WO2011127255 orWO2008103276, each of which is herein incorporated by reference in theirentirety. As a non-limiting example, modified RNA described herein maybe encapsulated in LNP formulations as described in WO2011127255 and/orWO2008103276; each of which is herein incorporated by reference in theirentirety.

In one embodiment, LNP formulations described herein may comprise apolycationic composition. As a non-limiting example, the polycationiccomposition may be selected from formula 1-60 of US Patent PublicationNo. US20050222064; herein incorporated by reference in its entirety. Inanother embodiment, the LNP formulations comprising a polycationiccomposition may be used for the delivery of the modified RNA describedherein in vivo and/or in vitro.

In one embodiment, the LNP formulations described herein mayadditionally comprise a permeability enhancer molecule. Non-limitingpermeability enhancer molecules are described in US Patent PublicationNo. US20050222064; herein incorporated by reference in its entirety.

In one embodiment, the pharmaceutical compositions may be formulated inliposomes such as, but not limited to, DiLa2 liposomes (Marina Biotech,Bothell, Wash.), SMARTICLES® (Marina Biotech, Bothell, Wash.), neutralDOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) based liposomes (e.g.,siRNA delivery for ovarian cancer (Landen et al. Cancer Biology &Therapy 2006 5(12)1708-1713)) and hyaluronan-coated liposomes (QuietTherapeutics, Israel).

Lipid nanoparticle formulations may be improved by replacing thecationic lipid with a biodegradable cationic lipid which is known as arapidly eliminated lipid nanoparticle (reLNP). Ionizable cationiclipids, such as, but not limited to, DLinDMA, DLin-KC2-DMA, andDLin-MC3-DMA, have been shown to accumulate in plasma and tissues overtime and may be a potential source of toxicity. The rapid metabolism ofthe rapidly eliminated lipids can improve the tolerability andtherapeutic index of the lipid nanoparticles by an order of magnitudefrom a 1 mg/kg dose to a 10 mg/kg dose in rat. Inclusion of anenzymatically degraded ester linkage can improve the degradation andmetabolism profile of the cationic component, while still maintainingthe activity of the reLNP formulation. The ester linkage can beinternally located within the lipid chain or it may be terminallylocated at the terminal end of the lipid chain. The internal esterlinkage may replace any carbon in the lipid chain.

In one embodiment, the internal ester linkage may be located on eitherside of the saturated carbon. Non-limiting examples of reLNPs include,

In one embodiment, an immune response may be elicited by delivering alipid nanoparticle which may include a nanospecies, a polymer and animmunogen. (U.S. Publication No. 20120189700 and InternationalPublication No. WO2012099805; each of which is herein incorporated byreference in their entirety). The polymer may encapsulate thenanospecies or partially encapsulate the nanospecies.

Lipid nanoparticles may be engineered to alter the surface properties ofparticles so the lipid nanoparticles may penetrate the mucosal barrier.Mucus is located on mucosal tissue such as, but not limited to, oral(e.g., the buccal and esophageal membranes and tonsil tissue),ophthalmic, gastrointestinal (e.g., stomach, small intestine, largeintestine, colon, rectum), nasal, respiratory (e.g., nasal, pharyngeal,tracheal and bronchial membranes), genital (e.g., vaginal, cervical andurethral membranes). Nanoparticles larger than 10-200 nm which arepreferred for higher drug encapsulation efficiency and the ability toprovide the sustained delivery of a wide array of drugs have beenthought to be too large to rapidly diffuse through mucosal barriers.Mucus is continuously secreted, shed, discarded or digested and recycledso most of the trapped particles may be removed from the mucosla tissuewithin seconds or within a few hours. Large polymeric nanoparticles (200nm-500 nm in diameter) which have been coated densely with a lowmolecular weight polyethylene glycol (PEG) diffused through mucus only 4to 6-fold lower than the same particles diffusing in water (Lai et al.PNAS 2007 104(5):1482-487; Lai et al. Adv Drug Deliv Rev. 2009 61(2):158-171; each of which is herein incorporated by reference in theirentirety). The transport of nanoparticles may be determined using ratesof permeation and/or fluorescent microscopy techniques including, butnot limited to, fluorescence recovery after photobleaching (FRAP) andhigh resolution multiple particle tracking (MPT).

The lipid nanoparticle engineered to penetrate mucus may comprise apolymeric material (i.e. a polymeric core) and/or a polymer-vitaminconjugate and/or a tri-block co-polymer. The polymeric material mayinclude, but is not limited to, polyamines, polyethers, polyamides,polyesters, polycarbamates, polyureas, polycarbonates, poly(styrenes),polyimides, polysulfones, polyurethanes, polyacetylenes, polyethylenes,polyethyeneimines, polyisocyanates, polyacrylates, polymethacrylates,polyacrylonitriles, and polyarylates. The polymeric material may bebiodegradable and/or biocompatible. Non-limiting examples of specificpolymers include poly(caprolactone) (PCL), ethylene vinyl acetatepolymer (EVA), poly(lactic acid) (PLA), poly(L-lactic acid) (PLLA),poly(glycolic acid) (PGA), poly(lactic acid-co-glycolic acid) (PLGA),poly(L-lactic acid-co-glycolic acid) (PLLGA), poly(D,L-lactide) (PDLA),poly(L-lactide) (PLLA), poly(D,L-lactide-co-caprolactone),poly(D,L-lactide-co-caprolactone-co-glycolide),poly(D,L-lactide-co-PEO-co-D,L-lactide),poly(D,L-lactide-co-PPO-co-D,L-lactide), polyalkyl cyanoacralate,polyurethane, poly-L-lysine (PLL), hydroxypropyl methacrylate (HPMA),polyethyleneglycol, poly-L-glutamic acid, poly(hydroxy acids),polyanhydrides, polyorthoesters, poly(ester amides), polyamides,poly(ester ethers), polycarbonates, polyalkylenes such as polyethyleneand polypropylene, polyalkylene glycols such as poly(ethylene glycol)(PEG), polyalkylene oxides (PEO), polyalkylene terephthalates such aspoly(ethylene terephthalate), polyvinyl alcohols (PVA), polyvinylethers, polyvinyl esters such as poly(vinyl acetate), polyvinyl halidessuch as poly(vinyl chloride) (PVC), polyvinylpyrrolidone, polysiloxanes,polystyrene (PS), polyurethanes, derivatized celluloses such as alkylcelluloses, hydroxyalkyl celluloses, cellulose ethers, cellulose esters,nitro celluloses, hydroxypropylcellulose, carboxymethylcellulose,polymers of acrylic acids, such as poly(methyl(meth)acrylate) (PMMA),poly(ethyl(meth)acrylate), poly(butyl(meth)acrylate),poly(isobutyl(meth)acrylate), poly(hexyl(meth)acrylate),poly(isodecyl(meth)acrylate), poly(lauryl(meth)acrylate),poly(phenyl(meth)acrylate), poly(methyl acrylate), poly(isopropylacrylate), poly(isobutyl acrylate), poly(octadecyl acrylate) andcopolymers and mixtures thereof, polydioxanone and its copolymers,polyhydroxyalkanoates, polypropylene fumarate, polyoxymethylene,poloxamers, poly(ortho)esters, poly(butyric acid), poly(valeric acid),poly(lactide-co-caprolactone), and trimethylene carbonate,polyvinylpyrrolidone. The lipid nanoparticle may be coated or associatedwith a co-polymer such as, but not limited to, a block co-polymer, and(poly(ethylene glycol))-(poly(propylene oxide))-(poly(ethylene glycol))triblock copolymer (see US Publication 20120121718 and US Publication20100003337; each of which is herein incorporated by reference in theirentirety). The co-polymer may be a polymer that is generally regarded assafe (GRAS) and the formation of the lipid nanoparticle may be in such away that no new chemical entities are created. For example, the lipidnanoparticle may comprise poloxamers coating PLGA nanoparticles withoutforming new chemical entities which are still able to rapidly penetratehuman mucus (Yang et al. Angew. Chem. Int. Ed. 2011 50:2597-2600; hereinincorporated by reference in its entirety).

The vitamin of the polymer-vitamin conjugate may be vitamin E. Thevitamin portion of the conjugate may be substituted with other suitablecomponents such as, but not limited to, vitamin A, vitamin E, othervitamins, cholesterol, a hydrophobic moiety, or a hydrophobic componentof other surfactants (e.g., sterol chains, fatty acids, hydrocarbonchains and alkylene oxide chains).

The lipid nanoparticle engineered to penetrate mucus may include surfacealtering agents such as, but not limited to, mmRNA, anionic protein(e.g., bovine serum albumin), surfactants (e.g., cationic surfactantssuch as for example dimethyldioctadecyl-ammonium bromide), sugars orsugar derivatives (e.g., cyclodextrin), nucleic acids, polymers (e.g.,heparin, polyethylene glycol and poloxamer), mucolytic agents (e.g.,N-acetylcysteine, mugwort, bromelain, papain, clerodendrum,acetylcysteine, bromhexine, carbocisteine, eprazinone, mesna, ambroxol,sobrerol, domiodol, letosteine, stepronin, tiopronin, gelsolin, thymosin(34 dornase alfa, neltenexine, erdosteine) and various DNases includingrhDNase. The surface altering agent may be embedded or enmeshed in theparticle's surface or disposed (e.g., by coating, adsorption, covalentlinkage, or other process) on the surface of the lipid nanoparticle.(see US Publication 20100215580 and US Publication 20080166414; each ofwhich is herein incorporated by reference in their entirety).

The mucus penetrating lipid nanoparticles may comprise at least onemmRNA described herein. The mmRNA may be encapsulated in the lipidnanoparticle and/or disposed on the surface of the particle. The mmRNAmay be covalently coupled to the lipid nanoparticle. Formulations ofmucus penetrating lipid nanoparticles may comprise a plurality ofnanoparticles. Further, the formulations may contain particles which mayinteract with the mucus and alter the structural and/or adhesiveproperties of the surrounding mucus to decrease mucoadhesion which mayincrease the delivery of the mucus penetrating lipid nanoparticles tothe mucosal tissue.

In one embodiment, the polynucleotide, primary construct, or mmRNA isformulated as a lipoplex, such as, without limitation, the ATUPLEX™system, the DACC system, the DBTC system and other siRNA-lipoplextechnology from Silence Therapeutics (London, United Kingdom), STEMFECT™from STEMGENT® (Cambridge, Mass.), and polyethylenimine (PEI) orprotamine-based targeted and non-targeted delivery of nucleic acids(Aleku et al. Cancer Res. 2008 68:9788-9798; Strumberg et al. Int J ClinPharmacol Ther 2012 50:76-78; Santel et al., Gene Ther 200613:1222-1234; Santel et al., Gene Ther 2006 13:1360-1370; Gutbier etal., Pulm Pharmacol. Ther. 2010 23:334-344; Kaufmann et al. MicrovascRes 2010 80:286-293Weide et al. J Immunother. 2009 32:498-507; Weide etal. J Immunother. 2008 31:180-188; Pascolo Expert Opin. Biol. Ther.4:1285-1294; Fotin-Mleczek et al., 2011 J. Immunother. 34:1-15; Song etal., Nature Biotechnol. 2005, 23:709-717; Peer et al., Proc Natl AcadSci USA. 2007 6; 104:4095-4100; deFougerolles Hum Gene Ther. 200819:125-132; all of which are incorporated herein by reference in itsentirety).

In one embodiment such formulations may also be constructed orcompositions altered such that they passively or actively are directedto different cell types in vivo, including but not limited tohepatocytes, immune cells, tumor cells, endothelial cells, antigenpresenting cells, and leukocytes (Akinc et al. Mol Ther. 201018:1357-1364; Song et al., Nat Biotechnol. 2005 23:709-717; Judge etal., J Clin Invest. 2009 119:661-673; Kaufmann et al., Microvasc Res2010 80:286-293; Santel et al., Gene Ther 2006 13:1222-1234; Santel etal., Gene Ther 2006 13:1360-1370; Gutbier et al., Pulm Pharmacol. Ther.2010 23:334-344; Basha et al., Mol. Ther. 2011 19:2186-2200; Fenske andCullis, Expert Opin Drug Deliv. 2008 5:25-44; Peer et al., Science. 2008319:627-630; Peer and Lieberman, Gene Ther. 2011 18:1127-1133; all ofwhich are incorporated herein by reference in its entirety). One exampleof passive targeting of formulations to liver cells includes theDLin-DMA, DLin-KC2-DMA and MC3-based lipid nanoparticle formulationswhich have been shown to bind to apolipoprotein E and promote bindingand uptake of these formulations into hepatocytes in vivo (Akinc et al.Mol Ther. 2010 18:1357-1364; herein incorporated by reference in itsentirety). Formulations can also be selectively targeted throughexpression of different ligands on their surface as exemplified by, butnot limited by, folate, transferrin, N-acetylgalactosamine (GalNAc), andantibody targeted approaches (Kolhatkar et al., Curr Drug DiscovTechnol. 2011 8:197-206; Musacchio and Torchilin, Front Biosci. 201116:1388-1412; Yu et al., Mol Membr Biol. 2010 27:286-298; Patil et al.,Crit Rev Ther Drug Carrier Syst. 2008 25:1-61; Benoit et al.,Biomacromolecules. 2011 12:2708-2714Zhao et al., Expert Opin Drug Deliv.2008 5:309-319; Akinc et al., Mol Ther. 2010 18:1357-1364; Srinivasan etal., Methods Mol Biol. 2012 820:105-116; Ben-Arie et al., Methods MolBiol. 2012 757:497-507; Peer 2010 J Control Release. 20:63-68; Peer etal., Proc Natl Acad Sci USA. 2007 104:4095-4100; Kim et al., Methods MolBiol. 2011 721:339-353; Subramanya et al., Mol Ther. 2010 18:2028-2037;Song et al., Nat Biotechnol. 2005 23:709-717; Peer et al., Science. 2008319:627-630; Peer and Lieberman, Gene Ther. 2011 18:1127-1133; all ofwhich are incorporated herein by reference in its entirety).

In one embodiment, the polynucleotide, primary construct, or mmRNA isformulated as a solid lipid nanoparticle. A solid lipid nanoparticle(SLN) may be spherical with an average diameter between 10 to 1000 nm.SLN possess a solid lipid core matrix that can solubilize lipophilicmolecules and may be stabilized with surfactants and/or emulsifiers. Ina further embodiment, the lipid nanoparticle may be a self-assemblylipid-polymer nanoparticle (see Zhang et al., ACS Nano, 2008, 2 (8), pp1696-1702; herein incorporated by reference in its entirety).

Liposomes, lipoplexes, or lipid nanoparticles may be used to improve theefficacy of polynucleotide, primary construct, or mmRNA directed proteinproduction as these formulations may be able to increase celltransfection by the polynucleotide, primary construct, or mmRNA; and/orincrease the translation of encoded protein. One such example involvesthe use of lipid encapsulation to enable the effective systemic deliveryof polyplex plasmid DNA (Heyes et al., Mol Ther. 2007 15:713-720; hereinincorporated by reference in its entirety). The liposomes, lipoplexes,or lipid nanoparticles may also be used to increase the stability of thepolynucleotide, primary construct, or mmRNA.

In one embodiment, the polynucleotides, primary constructs, and/or themmRNA of the present invention can be formulated for controlled releaseand/or targeted delivery. As used herein, “controlled release” refers toa pharmaceutical composition or compound release profile that conformsto a particular pattern of release to effect a therapeutic outcome. Inone embodiment, the polynucleotides, primary constructs or the mmRNA maybe encapsulated into a delivery agent described herein and/or known inthe art for controlled release and/or targeted delivery. As used herein,the term “encapsulate” means to enclose, surround or encase. As itrelates to the formulation of the compounds of the invention,encapsulation may be substantial, complete or partial. The term“substantially encapsulated” means that at least greater than 50, 60,70, 80, 85, 90, 95, 96, 97, 98, 99, 99.9, 99.9 or greater than 99.999%of the pharmaceutical composition or compound of the invention may beenclosed, surrounded or encased within the delivery agent. “Partiallyencapsulation” means that less than 10, 10, 20, 30, 40 50 or less of thepharmaceutical composition or compound of the invention may be enclosed,surrounded or encased within the delivery agent. Advantageously,encapsulation may be determined by measuring the escape or the activityof the pharmaceutical composition or compound of the invention usingfluorescence and/or electron micrograph. For example, at least 1, 5, 10,20, 30, 40, 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99, 99.9, 99.99 orgreater than 99.99% of the pharmaceutical composition or compound of theinvention are encapsulated in the delivery agent.

In another embodiment, the polynucleotides, primary constructs, or themmRNA may be encapsulated into a lipid nanoparticle or a rapidlyeliminating lipid nanoparticle and the lipid nanoparticles or a rapidlyeliminating lipid nanoparticle may then be encapsulated into a polymer,hydrogel and/or surgical sealant described herein and/or known in theart. As a non-limiting example, the polymer, hydrogel or surgicalsealant may be PLGA, ethylene vinyl acetate (EVAc), poloxamer, GELSITE®(Nanotherapeutics, Inc. Alachua, Fla.), HYLENEX® (Halozyme Therapeutics,San Diego Calif.), surgical sealants such as fibrinogen polymers(Ethicon Inc. Cornelia, Ga.), TISSELL® (Baxter International, IncDeerfield, Ill.), PEG-based sealants, and COSEAL® (Baxter International,Inc Deerfield, Ill.).

In one embodiment, the lipid nanoparticle may be encapsulated into anypolymer or hydrogel known in the art which may form a gel when injectedinto a subject. As another non-limiting example, the lipid nanoparticlemay be encapsulated into a polymer matrix which may be biodegradable.

In one embodiment, the polynucleotide, primary construct, or mmRNAformulation for controlled release and/or targeted delivery may alsoinclude at least one controlled release coating. Controlled releasecoatings include, but are not limited to, OPADRY®,polyvinylpyrrolidone/vinyl acetate copolymer, polyvinylpyrrolidone,hydroxypropyl methylcellulose, hydroxypropyl cellulose, hydroxyethylcellulose, EUDRAGIT RL®, EUDRAGIT RS® and cellulose derivatives such asethylcellulose aqueous dispersions (AQUACOAT® and SURELEASE®).

In one embodiment, the controlled release and/or targeted deliveryformulation may comprise at least one degradable polyester which maycontain polycationic side chains. Degradeable polyesters include, butare not limited to, poly(serine ester), poly(L-lactide-co-L-lysine),poly(4-hydroxy-L-proline ester), and combinations thereof. In anotherembodiment, the degradable polyesters may include a PEG conjugation toform a PEGylated polymer.

In one embodiment, the polynucleotides, primary constructs, and/or themmRNA of the present invention may be encapsulated in a therapeuticnanoparticle. Therapeutic nanoparticles may be formulated by methodsdescribed herein and known in the art such as, but not limited to,International Pub Nos. WO2010005740, WO2010030763, WO2010005721,WO2010005723, WO2012054923, US Pub. Nos. US20110262491, US20100104645,US20100087337, US20100068285, US20110274759, US20100068286, and U.S.Pat. No. 8,206,747; each of which is herein incorporated by reference intheir entirety. In another embodiment, therapeutic polymer nanoparticlesmay be identified by the methods described in US Pub No. US20120140790,herein incorporated by reference in its entirety.

In one embodiment, the therapeutic nanoparticle of may be formulated forsustained release. As used herein, “sustained release” refers to apharmaceutical composition or compound that conforms to a release rateover a specific period of time. The period of time may include, but isnot limited to, hours, days, weeks, months and years. As a non-limitingexample, the sustained release nanoparticle may comprise a polymer and atherapeutic agent such as, but not limited to, the polynucleotides,primary constructs, and mmRNA of the present invention (seeInternational Pub No. 2010075072 and US Pub No. US20100216804 andUS20110217377, each of which is herein incorporated by reference intheir entirety).

In one embodiment, the therapeutic nanoparticles may be formulated to betarget specific. As a non-limiting example, the therapeuticnanoparticles may include a corticosteroid (see International Pub. No.WO2011084518). In one embodiment, the therapeutic nanoparticles may beformulated to be cancer specific. As a non-limiting example, thetherapeutic nanoparticles may be formulated in nanoparticles describedin International Pub No. WO2008121949, WO2010005726, WO2010005725,WO2011084521 and US Pub No. US20100069426, US20120004293 andUS20100104655, each of which is herein incorporated by reference intheir entirety.

In one embodiment, the nanoparticles of the present invention maycomprise a polymeric matrix. As a non-limiting example, the nanoparticlemay comprise two or more polymers such as, but not limited to,polyethylenes, polycarbonates, polyanhydrides, polyhydroxyacids,polypropylfumerates, polycaprolactones, polyamides, polyacetals,polyethers, polyesters, poly(orthoesters), polycyanoacrylates, polyvinylalcohols, polyurethanes, polyphosphazenes, polyacrylates,polymethacrylates, polycyanoacrylates, polyureas, polystyrenes,polyamines, polylysine, poly(ethylene imine), poly(serine ester),poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline ester) orcombinations thereof.

In one embodiment, the diblock copolymer may include PEG in combinationwith a polymer such as, but not limited to, polyethylenes,polycarbonates, polyanhydrides, polyhydroxyacids, polypropylfumerates,polycaprolactones, polyamides, polyacetals, polyethers, polyesters,poly(orthoesters), polycyanoacrylates, polyvinyl alcohols,polyurethanes, polyphosphazenes, polyacrylates, polymethacrylates,polycyanoacrylates, polyureas, polystyrenes, polyamines, polylysine,poly(ethylene imine), poly(serine ester), poly(L-lactide-co-L-lysine),poly(4-hydroxy-L-proline ester) or combinations thereof.

In one embodiment, the therapeutic nanoparticle comprises a diblockcopolymer. As a non-limiting example the therapeutic nanoparticlecomprises a PLGA-PEG block copolymer (see US Pub. No. US20120004293 andU.S. Pat. No. 8,236,330, herein incorporated by reference in theirentireties). In another non-limiting example, the therapeuticnanoparticle is a stealth nanoparticle comprising a diblock copolymer ofPEG and PLA or PEG and PLGA (see U.S. Pat. No. 8,246,968, each of whichis herein incorporated by reference in its entirety).

In one embodiment, the therapeutic nanoparticle may comprise at leastone acrylic polymer. Acrylic polymers include but are not limited to,acrylic acid, methacrylic acid, acrylic acid and methacrylic acidcopolymers, methyl methacrylate copolymers, ethoxyethyl methacrylates,cyanoethyl methacrylate, amino alkyl methacrylate copolymer,poly(acrylic acid), poly(methacrylic acid), polycyanoacrylates andcombinations thereof.

In one embodiment, the therapeutic nanoparticles may comprise at leastone cationic polymer described herein and/or known in the art.

In one embodiment, the therapeutic nanoparticles may comprise at leastone amine-containing polymer such as, but not limited to polylysine,polyethylene imine, poly(amidoamine) dendrimers and combinationsthereof.

In one embodiment, the therapeutic nanoparticles may comprise at leastone degradable polyester which may contain polycationic side chains.Degradeable polyesters include, but are not limited to, poly(serineester), poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline ester),and combinations thereof. In another embodiment, the degradablepolyesters may include a PEG conjugation to form a PEGylated polymer.

In another embodiment, the therapeutic nanoparticle may include aconjugation of at least one targeting ligand.

In one embodiment, the therapeutic nanoparticle may be formulated in anaqueous solution which may be used to target cancer (see InternationalPub No. WO2011084513 and US Pub No. US20110294717, each of which isherein incorporated by reference in their entirety).

In one embodiment, the polynucleotides, primary constructs, or mmRNA maybe encapsulated in, linked to and/or associated with syntheticnanocarriers. The synthetic nanocarriers may be formulated using methodsknown in the art and/or described herein. As a non-limiting example, thesynthetic nanocarriers may be formulated by the methods described inInternational Pub Nos. WO2010005740, WO2010030763 and US Pub. Nos.US20110262491, US20100104645 and US20100087337, each of which is hereinincorporated by reference in their entirety. In another embodiment, thesynthetic nanocarrier formulations may be lyophilized by methodsdescribed in International Pub. No. WO2011072218 and U.S. Pat. No.8,211,473; each of which is herein incorporated by reference in theirentireties.

In one embodiment, the synthetic nanocarriers may contain reactivegroups to release the polynucleotides, primary constructs and/or mmRNAdescribed herein (see International Pub. No. WO20120952552 and US PubNo. US20120171229, each of which is herein incorporated by reference intheir entirety).

In one embodiment, the synthetic nanocarriers may contain animmunostimulatory agent to enhance the immune response from delivery ofthe synthetic nanocarrier. As a non-limiting example, the syntheticnanocarrier may comprise a Th1 immunostimulatory agent which may enhancea Th1-based response of the immune system (see International Pub No.WO2010123569 and US Pub. No. US20110223201, each of which is hereinincorporated by reference in its entirety).

In one embodiment, the synthetic nanocarriers may be formulated fortargeted release. In one embodiment, the synthetic nanocarrier isformulated to release the polynucleotides, primary constructs and/ormmRNA at a specified pH and/or after a desired time interval. As anon-limiting example, the synthetic nanoparticle may be formulated torelease the polynucleotides, primary constructs and/or mmRNA after 24hours and/or at a pH of 4.5 (see International Pub. Nos. WO2010138193and WO2010138194 and US Pub Nos. US20110020388 and US20110027217, eachof which is herein incorporated by reference in their entireties).

In one embodiment, the synthetic nanocarriers may be formulated forcontrolled and/or sustained release of the polynucleotides, primaryconstructs and/or mmRNA described herein. As a non-limiting example, thesynthetic nanocarriers for sustained release may be formulated bymethods known in the art, described herein and/or as described inInternational Pub No. WO2010138192 and US Pub No. 20100303850, each ofwhich is herein incorporated by reference in their entireties.

Polymers, Biodegradable Nanoparticles, and Core-Shell Nanoparticles

The polynucleotide, primary construct, and mmRNA of the invention can beformulated using natural and/or synthetic polymers. Non-limitingexamples of polymers which may be used for delivery include, but are notlimited to, Dynamic POLYCONJUGATE′ formulations from MIRUS® Bio(Madison, Wis.) and Roche Madison (Madison, Wis.), PHASERX′ polymerformulations such as, without limitation, SMARTT POLYMER TECHNOLOGY™(Seattle, Wash.), DMRI/DOPE, poloxamer, VAXFECTIN® adjuvant from Vical(San Diego, Calif.), chitosan, cyclodextrin from Calando Pharmaceuticals(Pasadena, Calif.), dendrimers and poly(lactic-co-glycolic acid) (PLGA)polymers. RONDEL™ (RNAi/Oligonucleotide Nanoparticle Delivery) polymers(Arrowhead Research Corporation, Pasadena, Calif.) and pH responsiveco-block polymers such as, but not limited to, PHASERX™ (Seattle,Wash.).

A non-limiting example of PLGA formulations include, but are not limitedto, PLGA injectable depots (e.g., ELIGARD® which is formed by dissolvingPLGA in 66% N-methyl-2-pyrrolidone (NMP) and the remainder being aqueoussolvent and leuprolide. Once injected, the PLGA and leuprolide peptideprecipitates into the subcutaneous space).

Many of these polymer approaches have demonstrated efficacy indelivering oligonucleotides in vivo into the cell cytoplasm (reviewed indeFougerolles Hum Gene Ther. 2008 19:125-132; herein incorporated byreference in its entirety). Two polymer approaches that have yieldedrobust in vivo delivery of nucleic acids, in this case with smallinterfering RNA (siRNA), are dynamic polyconjugates andcyclodextrin-based nanoparticles. The first of these delivery approachesuses dynamic polyconjugates and has been shown in vivo in mice toeffectively deliver siRNA and silence endogenous target mRNA inhepatocytes (Rozema et al., Proc Natl Acad Sci USA. 2007104:12982-12887). This particular approach is a multicomponent polymersystem whose key features include a membrane-active polymer to whichnucleic acid, in this case siRNA, is covalently coupled via a disulfidebond and where both PEG (for charge masking) and N-acetylgalactosamine(for hepatocyte targeting) groups are linked via pH-sensitive bonds(Rozema et al., Proc Natl Acad Sci USA. 2007 104:12982-12887). Onbinding to the hepatocyte and entry into the endosome, the polymercomplex disassembles in the low-pH environment, with the polymerexposing its positive charge, leading to endosomal escape andcytoplasmic release of the siRNA from the polymer. Through replacementof the N-acetylgalactosamine group with a mannose group, it was shownone could alter targeting from asialoglycoprotein receptor-expressinghepatocytes to sinusoidal endothelium and Kupffer cells. Another polymerapproach involves using transferrin-targeted cyclodextrin-containingpolycation nanoparticles. These nanoparticles have demonstrated targetedsilencing of the EWS-FLI1 gene product in transferrinreceptor-expressing Ewing's sarcoma tumor cells (Hu-Lieskovan et al.,Cancer Res. 2005 65: 8984-8982) and siRNA formulated in thesenanoparticles was well tolerated in non-human primates (Heidel et al.,Proc Natl Acad Sci USA 2007 104:5715-21). Both of these deliverystrategies incorporate rational approaches using both targeted deliveryand endosomal escape mechanisms.

The polymer formulation can permit the sustained or delayed release ofthe polynucleotide, primary construct, or mmRNA (e.g., followingintramuscular or subcutaneous injection). The altered release profilefor the polynucleotide, primary construct, or mmRNA can result in, forexample, translation of an encoded protein over an extended period oftime. The polymer formulation may also be used to increase the stabilityof the polynucleotide, primary construct, or mmRNA. Biodegradablepolymers have been previously used to protect nucleic acids other thanmmRNA from degradation and been shown to result in sustained release ofpayloads in vivo (Rozema et al., Proc Natl Acad Sci USA. 2007104:12982-12887; Sullivan et al., Expert Opin Drug Deliv. 20107:1433-1446; Convertine et al., Biomacromolecules. 2010 Oct. 1; Chu etal., Acc Chem Res. 2012 Jan. 13; Manganiello et al., Biomaterials. 201233:2301-2309; Benoit et al., Biomacromolecules. 2011 12:2708-2714;Singha et al., Nucleic Acid Ther. 2011 2:133-147; deFougerolles Hum GeneTher. 2008 19:125-132; Schaffert and Wagner, Gene Ther. 200816:1131-1138; Chaturvedi et al., Expert Opin Drug Deliv. 20118:1455-1468; Davis, Mol Pharm. 2009 6:659-668; Davis, Nature 2010464:1067-1070; each of which is herein incorporated by reference in itsentirety).

In one embodiment, the pharmaceutical compositions may be sustainedrelease formulations. In a further embodiment, the sustained releaseformulations may be for subcutaneous delivery. Sustained releaseformulations may include, but are not limited to, PLGA microspheres,ethylene vinyl acetate (EVAc), poloxamer, GELSITE® (Nanotherapeutics,Inc. Alachua, Fla.), HYLENEX® (Halozyme Therapeutics, San Diego Calif.),surgical sealants such as fibrinogen polymers (Ethicon Inc. Cornelia,Ga.), TISSELL® (Baxter International, Inc Deerfield, Ill.), PEG-basedsealants, and COSEAL® (Baxter International, Inc Deerfield, Ill.).

As a non-limiting example modified mRNA may be formulated in PLGAmicrospheres by preparing the PLGA microspheres with tunable releaserates (e.g., days and weeks) and encapsulating the modified mRNA in thePLGA microspheres while maintaining the integrity of the modified mRNAduring the encapsulation process. EVAc are non-biodegradeable,biocompatible polymers which are used extensively in pre-clinicalsustained release implant applications (e.g., extended release productsOcusert a pilocarpine ophthalmic insert for glaucoma or progestasert asustained release progesterone intrauterine device; transdermal deliverysystems Testoderm, Duragesic and Selegiline; catheters). Poloxamer F-407NF is a hydrophilic, non-ionic surfactant triblock copolymer ofpolyoxyethylene-polyoxypropylene-polyoxyethylene having a low viscosityat temperatures less than 5° C. and forms a solid gel at temperaturesgreater than 15° C. PEG-based surgical sealants comprise two syntheticPEG components mixed in a delivery device which can be prepared in oneminute, seals in 3 minutes and is reabsorbed within 30 days. GELSITE®and natural polymers are capable of in-situ gelation at the site ofadministration. They have been shown to interact with protein andpeptide therapeutic candidates through ionic interaction to provide astabilizing effect.

Polymer formulations can also be selectively targeted through expressionof different ligands as exemplified by, but not limited by, folate,transferrin, and N-acetylgalactosamine (GalNAc) (Benoit et al.,Biomacromolecules. 2011 12:2708-2714; Rozema et al., Proc Natl Acad SciUSA. 2007 104:12982-12887; Davis, Mol Pharm. 2009 6:659-668; Davis,Nature 2010 464:1067-1070; each of which is herein incorporated byreference in its entirety).

The polynucleotides, primary constructs and/or mmRNA of the inventionmay be formulated with or in a polymeric compound. The polymer mayinclude at least one polymer such as, but not limited to, polyethenes,polyethylene glycol (PEG), poly(l-lysine)(PLL), PEG grafted to PLL,cationic lipopolymer, biodegradable cationic lipopolymer,polyethyleneimine (PEI), cross-linked branched poly(alkylene imines), apolyamine derivative, a modified poloxamer, a biodegradable polymer,biodegradable block copolymer, biodegradable random copolymer,biodegradable polyester copolymer, biodegradable polyester blockcopolymer, biodegradable polyester block random copolymer, linearbiodegradable copolymer, poly[α-(4-aminobutyl)-L-glycolic acid) (PAGA),biodegradable cross-linked cationic multi-block copolymers,polycarbonates, polyanhydrides, polyhydroxyacids, polypropylfumerates,polycaprolactones, polyamides, polyacetals, polyethers, polyesters,poly(orthoesters), polycyanoacrylates, polyvinyl alcohols,polyurethanes, polyphosphazenes, polyacrylates, polymethacrylates,polycyanoacrylates, polyureas, polystyrenes, polyamines, polylysine,poly(ethylene imine), poly(serine ester), poly(L-lactide-co-L-lysine),poly(4-hydroxy-L-proline ester), acrylic polymers, amine-containingpolymers or combinations thereof.

As a non-limiting example, the polynucleotides, primary constructsand/or mmRNA of the invention may be formulated with the polymericcompound of PEG grafted with PLL as described in U.S. Pat. No. 6,177,274herein incorporated by reference in its entirety. The formulation may beused for transfecting cells in vitro or for in vivo delivery of thepolynucleotides, primary constructs and/or mmRNA. In another example,the polynucleotides, primary constructs and/or mmRNA may be suspended ina solution or medium with a cationic polymer, in a dry pharmaceuticalcomposition or in a solution that is capable of being dried as describedin U.S. Pub. Nos. 20090042829 and 20090042825 each of which are hereinincorporated by reference in their entireties.

As another non-limiting example the polynucleotides, primary constructsor mmRNA of the invention may be formulated with a PLGA-PEG blockcopolymer (see US Pub. No. US20120004293 and U.S. Pat. No. 8,236,330,each of which is herein incorporated by reference in their entireties).As a non-limiting example, the polynucleotides, primary constructs ormmRNA of the invention may be formulated with a diblock copolymer of PEGand PLA or PEG and PLGA (see U.S. Pat. No. 8,246,968, hereinincorporated by reference in its entirety).

A polyamine derivative may be used to deliver nucleic acids or to treatand/or prevent a disease or to be included in an implantable orinjectable device (U.S. Pub. No. 20100260817 herein incorporated byreference in its entirety). As a non-limiting example, a pharmaceuticalcomposition may include the modified nucleic acids and mmRNA and thepolyamine derivative described in U.S. Pub. No. 20100260817 (thecontents of which are incorporated herein by reference in its entirety.

The polynucleotides, primary constructs or mmRNA of the invention may beformulated with at least one acrylic polymer. Acrylic polymers includebut are not limited to, acrylic acid, methacrylic acid, acrylic acid andmethacrylic acid copolymers, methyl methacrylate copolymers, ethoxyethylmethacrylates, cyanoethyl methacrylate, amino alkyl methacrylatecopolymer, poly(acrylic acid), poly(methacrylic acid),polycyanoacrylates and combinations thereof.

In one embodiment, the polynucleotides, primary constructs or mmRNA ofthe present invention may be formulated with at least one polymerdescribed in International Publication Nos. WO2011115862, WO2012082574and WO2012068187, each of which is herein incorporated by reference intheir entireties. In another embodiment the polynucleotides, primaryconstructs or mmRNA of the present invention may be formulated with apolymer of formula Z as described in WO2011115862, herein incorporatedby reference in its entirety. In yet another embodiment, thepolynucleotides, primary constructs or mmRNA may be formulated with apolymer of formula Z, Z′ or Z″ as described in WO2012082574 orWO2012068187, each of which are herein incorporated by reference intheir entireties. The polymers formulated with the modified RNA of thepresent invention may be synthesized by the methods described inWO2012082574 or WO2012068187, each of which is herein incorporated byreference in their entireties.

Formulations of polynucleotides, primary constructs or mmRNA of theinvention may include at least one amine-containing polymer such as, butnot limited to polylysine, polyethylene imine, poly(amidoamine)dendrimers or combinations thereof.

For example, the polynucleotides, primary constructs and/or mmRNA of theinvention may be formulated in a pharmaceutical compound including apoly(alkylene imine), a biodegradable cationic lipopolymer, abiodegradable block copolymer, a biodegradable polymer, or abiodegradable random copolymer, a biodegradable polyester blockcopolymer, a biodegradable polyester polymer, a biodegradable polyesterrandom copolymer, a linear biodegradable copolymer, PAGA, abiodegradable cross-linked cationic multi-block copolymer orcombinations thereof. The biodegradable cationic lipopolymer may be mademy methods known in the art and/or described in U.S. Pat. No. 6,696,038,U.S. App. Nos. 20030073619 and 20040142474 each of which is hereinincorporated by reference in their entireties. The poly(alkylene imine)may be made using methods known in the art and/or as described in U.S.Pub. No. 20100004315, herein incorporated by reference in its entirety.The biodegradable polymer, biodegradable block copolymer, thebiodegradable random copolymer, biodegradable polyester block copolymer,biodegradable polyester polymer, or biodegradable polyester randomcopolymer may be made using methods known in the art and/or as describedin U.S. Pat. Nos. 6,517,869 and 6,267,987, the contents of which areeach incorporated herein by reference in its entirety. The linearbiodegradable copolymer may be made using methods known in the artand/or as described in U.S. Pat. No. 6,652,886. The PAGA polymer may bemade using methods known in the art and/or as described in U.S. Pat. No.6,217,912 herein incorporated by reference in its entirety. The PAGApolymer may be copolymerized to form a copolymer or block copolymer withpolymers such as but not limited to, poly-L-lysine, polyargine,polyornithine, histones, avidin, protamines, polylactides andpoly(lactide-co-glycolides). The biodegradable cross-linked cationicmulti-block copolymers may be made my methods known in the art and/or asdescribed in U.S. Pat. No. 8,057,821 or U.S. Pub. No. 2012009145 each ofwhich is herein incorporated by reference in their entireties. Forexample, the multi-block copolymers may be synthesized using linearpolyethyleneimine (LPEI) blocks which have distinct patterns as comparedto branched polyethyleneimines. Further, the composition orpharmaceutical composition may be made by the methods known in the art,described herein, or as described in U.S. Pub. No. 20100004315 or U.S.Pat. Nos. 6,267,987 and 6,217,912 each of which is herein incorporatedby reference in their entireties.

The polynucleotides, primary constructs, and mmRNA of the invention maybe formulated with at least one degradable polyester which may containpolycationic side chains. Degradeable polyesters include, but are notlimited to, poly(serine ester), poly(L-lactide-co-L-lysine),poly(4-hydroxy-L-proline ester), and combinations thereof. In anotherembodiment, the degradable polyesters may include a PEG conjugation toform a PEGylated polymer.

In one embodiment, the polymers described herein may be conjugated to alipid-terminating PEG. As a non-limiting example, PLGA may be conjugatedto a lipid-terminating PEG forming PLGA-DSPE-PEG. As anothernon-limiting example, PEG conjugates for use with the present inventionare described in International Publication No. WO2008103276, hereinincorporated by reference in its entirety.

In one embodiment, the polynucleotides, primary constructs and/or mmRNAdescribed herein may be conjugated with another compound. Non-limitingexamples of conjugates are described in U.S. Pat. Nos. 7,964,578 and7,833,992, each of which are herein incorporated by reference in theirentireties. In another embodiment, the polynucleotides, primaryconstructs and/or mmRNA of the present invention may be conjugated withconjugates of formula 1-122 as described in U.S. Pat. Nos. 7,964,578 and7,833,992, each of which are herein incorporated by reference in theirentireties.

As described in U.S. Pub. No. 20100004313, herein incorporated byreference in its entirety, a gene delivery composition may include anucleotide sequence and a poloxamer. For example, the polynucleotide,primary construct and/or mmRNA of the present invention may be used in agene delivery composition with the poloxamer described in U.S. Pub. No.20100004313.

In one embodiment, the polymer formulation of the present invention maybe stabilized by contacting the polymer formulation, which may include acationic carrier, with a cationic lipopolymer which may be covalentlylinked to cholesterol and polyethylene glycol groups. The polymerformulation may be contacted with a cationic lipopolymer using themethods described in U.S. Pub. No. 20090042829 herein incorporated byreference in its entirety. The cationic carrier may include, but is notlimited to, polyethylenimine, poly(trimethylenimine),poly(tetramethylenimine), polypropylenimine, aminoglycoside-polyamine,dideoxy-diamino-b-cyclodextrin, spermine, spermidine,poly(2-dimethylamino)ethyl methacrylate, poly(lysine), poly(histidine),poly(arginine), cationized gelatin, dendrimers, chitosan,1,2-Dioleoyl-3-Trimethylammonium-Propane(DOTAP),N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA),1-[2-(oleoyloxy)ethyl]-2-oleyl-3-(2-hydroxyethyl)imidazolinium chloride(DOTIM),2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminiumtrifluoroacetate (DOSPA),3B-[N—(N′,N′-Dimethylaminoethane)-carbamoyl]Cholesterol Hydrochloride(DC-Cholesterol HCl) diheptadecylamidoglycyl spermidine (DOGS),N,N-distearyl-N,N-dimethylammonium bromide (DDAB),N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammoniumbromide (DMRIE), N,N-dioleyl-N,N-dimethylammonium chloride DODAC) andcombinations thereof.

The polynucleotide, primary construct, and mmRNA of the invention canalso be formulated as a nanoparticle using a combination of polymers,lipids, and/or other biodegradable agents, such as, but not limited to,calcium phosphate. Components may be combined in a core-shell, hybrid,and/or layer-by-layer architecture, to allow for fine-tuning of thenanoparticle so to delivery of the polynucleotide, primary construct andmmRNA may be enhanced (Wang et al., Nat Mater. 2006 5:791-796; Fuller etal., Biomaterials. 2008 29:1526-1532; DeKoker et al., Adv Drug DelivRev. 2011 63:748-761; Endres et al., Biomaterials. 2011 32:7721-7731; Suet al., Mol Pharm. 2011 Jun. 6; 8(3):774-87; herein incorporated byreference in its entirety).

Biodegradable calcium phosphate nanoparticles in combination with lipidsand/or polymers have been shown to deliver polynucleotides, primaryconstructs and mmRNA in vivo. In one embodiment, a lipid coated calciumphosphate nanoparticle, which may also contain a targeting ligand suchas anisamide, may be used to deliver the polynucleotide, primaryconstruct and mmRNA of the present invention. For example, toeffectively deliver siRNA in a mouse metastatic lung model a lipidcoated calcium phosphate nanoparticle was used (Li et al., J Contr Rel.2010 142: 416-421; Li et al., J Contr Rel. 2012 158:108-114; Yang etal., Mol Ther. 2012 20:609-615). This delivery system combines both atargeted nanoparticle and a component to enhance the endosomal escape,calcium phosphate, in order to improve delivery of the siRNA.

In one embodiment, calcium phosphate with a PEG-polyanion blockcopolymer may be used to delivery polynucleotides, primary constructsand mmRNA (Kazikawa et al., J Contr Rel. 2004 97:345-356; Kazikawa etal., J Contr Rel. 2006 111:368-370).

In one embodiment, a PEG-charge-conversional polymer (Pitella et al.,Biomaterials. 2011 32:3106-3114) may be used to form a nanoparticle todeliver the polynucleotides, primary constructs and mmRNA of the presentinvention. The PEG-charge-conversional polymer may improve upon thePEG-polyanion block copolymers by being cleaved into a polycation atacidic pH, thus enhancing endosomal escape.

The use of core-shell nanoparticles has additionally focused on ahigh-throughput approach to synthesize cationic cross-linked nanogelcores and various shells (Siegwart et al., Proc Natl Acad Sci USA. 2011108:12996-13001). The complexation, delivery, and internalization of thepolymeric nanoparticles can be precisely controlled by altering thechemical composition in both the core and shell components of thenanoparticle. For example, the core-shell nanoparticles may efficientlydeliver siRNA to mouse hepatocytes after they covalently attachcholesterol to the nanoparticle.

In one embodiment, a hollow lipid core comprising a middle PLGA layerand an outer neutral lipid layer containing PEG may be used to deliveryof the polynucleotide, primary construct and mmRNA of the presentinvention. As a non-limiting example, in mice bearing aluciferease-expressing tumor, it was determined that thelipid-polymer-lipid hybrid nanoparticle significantly suppressedluciferase expression, as compared to a conventional lipoplex (Shi etal, Angew Chem Int Ed. 2011 50:7027-7031).

Peptides and Proteins

The polynucleotide, primary construct, and mmRNA of the invention can beformulated with peptides and/or proteins in order to increasetransfection of cells by the polynucleotide, primary construct, ormmRNA. In one embodiment, peptides such as, but not limited to, cellpenetrating peptides and proteins and peptides that enable intracellulardelivery may be used to deliver pharmaceutical formulations. Anon-limiting example of a cell penetrating peptide which may be usedwith the pharmaceutical formulations of the present invention includes acell-penetrating peptide sequence attached to polycations thatfacilitates delivery to the intracellular space, e.g., HIV-derived TATpeptide, penetratins, transportans, or hCT derived cell-penetratingpeptides (see, e.g., Caron et al., Mol. Ther. 3(3):310-8 (2001); Langel,Cell-Penetrating Peptides: Processes and Applications (CRC Press, BocaRaton Fla., 2002); El-Andaloussi et al., Curr. Pharm. Des.11(28):3597-611 (2003); and Deshayes et al., Cell. Mol. Life Sci.62(16):1839-49 (2005), all of which are incorporated herein byreference). The compositions can also be formulated to include a cellpenetrating agent, e.g., liposomes, which enhance delivery of thecompositions to the intracellular space. polynucleotides, primaryconstructs, and mmRNA of the invention may be complexed to peptidesand/or proteins such as, but not limited to, peptides and/or proteinsfrom Aileron Therapeutics (Cambridge, Mass.) and Permeon Biologics(Cambridge, Mass.) in order to enable intracellular delivery (Cronicanet al., ACS Chem. Biol. 2010 5:747-752; McNaughton et al., Proc. Natl.Acad. Sci. USA 2009 106:6111-6116; Sawyer, Chem Biol Drug Des. 200973:3-6; Verdine and Hilinski, Methods Enzymol. 2012; 503:3-33; all ofwhich are herein incorporated by reference in its entirety).

In one embodiment, the cell-penetrating polypeptide may comprise a firstdomain and a second domain. The first domain may comprise a superchargedpolypeptide. The second domain may comprise a protein-binding partner.As used herein, “protein-binding partner” includes, but are not limitedto, antibodies and functional fragments thereof, scaffold proteins, orpeptides. The cell-penetrating polypeptide may further comprise anintracellular binding partner for the protein-binding partner. Thecell-penetrating polypeptide may be capable of being secreted from acell where the polynucleotide, primary construct, or mmRNA may beintroduced.

Formulations of the including peptides or proteins may be used toincrease cell transfection by the polynucleotide, primary construct, ormmRNA, alter the biodistribution of the polynucleotide, primaryconstruct, or mmRNA (e.g., by targeting specific tissues or cell types),and/or increase the translation of encoded protein.

Cells

The polynucleotide, primary construct, and mmRNA of the invention can betransfected ex vivo into cells, which are subsequently transplanted intoa subject. As non-limiting examples, the pharmaceutical compositions mayinclude red blood cells to deliver modified RNA to liver and myeloidcells, virosomes to deliver modified RNA in virus-like particles (VLPs),and electroporated cells such as, but not limited to, from MAXCYTE®(Gaithersburg, Md.) and from ERYTECH® (Lyon, France) to deliver modifiedRNA. Examples of use of red blood cells, viral particles andelectroporated cells to deliver payloads other than mmRNA have beendocumented (Godfrin et al., Expert Opin Biol Ther. 2012 12:127-133; Fanget al., Expert Opin Biol Ther. 2012 12:385-389; Hu et al., Proc NatlAcad Sci USA. 2011 108:10980-10985; Lund et al., Pharm Res. 201027:400-420; Huckriede et al., J Liposome Res. 2007; 17:39-47; Cusi, HumVaccin. 2006 2:1-7; de Jonge et al., Gene Ther. 2006 13:400-411; all ofwhich are herein incorporated by reference in its entirety).

The polynucleotides, primary constructs and mmRNA may be delivered insynthetic VLPs synthesized by the methods described in International PubNo. WO2011085231 and US Pub No. 20110171248, each of which is hereinincorporated by reference in their entireties.

Cell-based formulations of the polynucleotide, primary construct, andmmRNA of the invention may be used to ensure cell transfection (e.g., inthe cellular carrier), alter the biodistribution of the polynucleotide,primary construct, or mmRNA (e.g., by targeting the cell carrier tospecific tissues or cell types), and/or increase the translation ofencoded protein.

A variety of methods are known in the art and suitable for introductionof nucleic acid into a cell, including viral and non-viral mediatedtechniques. Examples of typical non-viral mediated techniques include,but are not limited to, electroporation, calcium phosphate mediatedtransfer, nucleofection, sonoporation, heat shock, magnetofection,liposome mediated transfer, microinjection, microprojectile mediatedtransfer (nanoparticles), cationic polymer mediated transfer(DEAE-dextran, polyethylenimine, polyethylene glycol (PEG) and the like)or cell fusion.

The technique of sonoporation, or cellular sonication, is the use ofsound (e.g., ultrasonic frequencies) for modifying the permeability ofthe cell plasma membrane. Sonoporation methods are known to those in theart and are used to deliver nucleic acids in vivo (Yoon and Park, ExpertOpin Drug Deliv. 2010 7:321-330; Postema and Gilja, Curr PharmBiotechnol. 2007 8:355-361; Newman and Bettinger, Gene Ther. 200714:465-475; all herein incorporated by reference in their entirety).Sonoporation methods are known in the art and are also taught forexample as it relates to bacteria in US Patent Publication 20100196983and as it relates to other cell types in, for example, US PatentPublication 20100009424, each of which are incorporated herein byreference in their entirety.

Electroporation techniques are also well known in the art and are usedto deliver nucleic acids in vivo and clinically (Andre et al., Curr GeneTher. 2010 10:267-280; Chiarella et al., Curr Gene Ther. 201010:281-286; Hojman, Curr Gene Ther. 2010 10:128-138; all hereinincorporated by reference in their entirety). In one embodiment,polynucleotides, primary constructs or mmRNA may be delivered byelectroporation as described in Example 26.

Hyaluronidase

The intramuscular or subcutaneous localized injection of polynucleotide,primary construct, or mmRNA of the invention can include hyaluronidase,which catalyzes the hydrolysis of hyaluronan. By catalyzing thehydrolysis of hyaluronan, a constituent of the interstitial barrier,hyaluronidase lowers the viscosity of hyaluronan, thereby increasingtissue permeability (Frost, Expert Opin. Drug Deliv. (2007) 4:427-440;herein incorporated by reference in its entirety). It is useful to speedtheir dispersion and systemic distribution of encoded proteins producedby transfected cells. Alternatively, the hyaluronidase can be used toincrease the number of cells exposed to a polynucleotide, primaryconstruct, or mmRNA of the invention administered intramuscularly orsubcutaneously.

Nanoparticle Mimics

The polynucleotide, primary construct or mmRNA of the invention may beencapsulated within and/or absorbed to a nanoparticle mimic. Ananoparticle mimic can mimic the delivery function organisms orparticles such as, but not limited to, pathogens, viruses, bacteria,fungus, parasites, prions and cells. As a non-limiting example thepolynucleotide, primary construct or mmRNA of the invention may beencapsulated in a non-viron particle which can mimic the deliveryfunction of a virus (see International Pub. No. WO2012006376 hereinincorporated by reference in its entirety).

Nanotubes

The polynucleotides, primary constructs or mmRNA of the invention can beattached or otherwise bound to at least one nanotube such as, but notlimited to, rosette nanotubes, rosette nanotubes having twin bases witha linker, carbon nanotubes and/or single-walled carbon nanotubes, Thepolynucleotides, primary constructs or mmRNA may be bound to thenanotubes through forces such as, but not limited to, steric, ionic,covalent and/or other forces.

In one embodiment, the nanotube can release one or more polynucleotides,primary constructs or mmRNA into cells. The size and/or the surfacestructure of at least one nanotube may be altered so as to govern theinteraction of the nanotubes within the body and/or to attach or bind tothe polynucleotides, primary constructs or mmRNA disclosed herein. Inone embodiment, the building block and/or the functional groups attachedto the building block of the at least one nanotube may be altered toadjust the dimensions and/or properties of the nanotube. As anon-limiting example, the length of the nanotubes may be altered tohinder the nanotubes from passing through the holes in the walls ofnormal blood vessels but still small enough to pass through the largerholes in the blood vessels of tumor tissue.

In one embodiment, at least one nanotube may also be coated withdelivery enhancing compounds including polymers, such as, but notlimited to, polyethylene glycol. In another embodiment, at least onenanotube and/or the polynucleotides, primary constructs or mmRNA may bemixed with pharmaceutically acceptable excipients and/or deliveryvehicles.

In one embodiment, the polynucleotides, primary constructs or mmRNA areattached and/or otherwise bound to at least one rosette nanotube. Therosette nanotubes may be formed by a process known in the art and/or bythe process described in International Publication No. WO2012094304,herein incorporated by reference in its entirety. At least onepolynucleotide, primary construct and/or mmRNA may be attached and/orotherwise bound to at least one rosette nanotube by a process asdescribed in International Publication No. WO2012094304, hereinincorporated by reference in its entirety, where rosette nanotubes ormodules forming rosette nanotubes are mixed in aqueous media with atleast one polynucleotide, primary construct and/or mmRNA underconditions which may cause at least one polynucleotide, primaryconstruct or mmRNA to attach or otherwise bind to the rosette nanotubes.

Conjugates

The polynucleotides, primary constructs, and mmRNA of the inventioninclude conjugates, such as a polynucleotide, primary construct, ormmRNA covalently linked to a carrier or targeting group, or includingtwo encoding regions that together produce a fusion protein (e.g.,bearing a targeting group and therapeutic protein or peptide).

The conjugates of the invention include a naturally occurring substance,such as a protein (e.g., human serum albumin (HSA), low-densitylipoprotein (LDL), high-density lipoprotein (HDL), or globulin); ancarbohydrate (e.g., a dextran, pullulan, chitin, chitosan, inulin,cyclodextrin or hyaluronic acid); or a lipid. The ligand may also be arecombinant or synthetic molecule, such as a synthetic polymer, e.g., asynthetic polyamino acid, an oligonucleotide (e.g. an aptamer). Examplesof polyamino acids include polyamino acid is a polylysine (PLL), polyL-aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydridecopolymer, poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleicanhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA),polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane,poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, orpolyphosphazine. Example of polyamines include: polyethylenimine,polylysine (PLL), spermine, spermidine, polyamine,pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine,arginine, amidine, protamine, cationic lipid, cationic porphyrin,quaternary salt of a polyamine, or an alpha helical peptide.

Representative U.S. patents that teach the preparation of polynucleotideconjugates, particularly to RNA, include, but are not limited to, U.S.Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313;5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,591,584; 5,109,124;5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718;5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737;4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830;5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022;5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098;5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667;5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371;5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941; 6,294,664;6,320,017; 6,576,752; 6,783,931; 6,900,297; 7,037,646; each of which isherein incorporated by reference in their entireties.

In one embodiment, the conjugate of the present invention may functionas a carrier for the polynucleotides, primary constructs and/or mmRNA ofthe present invention. The conjugate may comprise a cationic polymersuch as, but not limited to, polyamine, polylysine, polyalkylenimine,and polyethylenimine which may be grafted to with poly(ethylene glycol).As a non-limiting example, the conjugate may be similar to the polymericconjugate and the method of synthesizing the polymeric conjugatedescribed in U.S. Pat. No. 6,586,524 herein incorporated by reference inits entirety.

The conjugates can also include targeting groups, e.g., a cell or tissuetargeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g.,an antibody, that binds to a specified cell type such as a kidney cell.A targeting group can be a thyrotropin, melanotropin, lectin,glycoprotein, surfactant protein A, Mucin carbohydrate, multivalentlactose, multivalent galactose, N-acetyl-galactosamine,N-acetyl-gulucosamine multivalent mannose, multivalent fucose,glycosylated polyaminoacids, multivalent galactose, transferrin,bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, asteroid, bile acid, folate, vitamin B12, biotin, an RGD peptide, an RGDpeptide mimetic or an aptamer.

Targeting groups can be proteins, e.g., glycoproteins, or peptides,e.g., molecules having a specific affinity for a co-ligand, orantibodies e.g., an antibody, that binds to a specified cell type suchas a cancer cell, endothelial cell, or bone cell. Targeting groups mayalso include hormones and hormone receptors. They can also includenon-peptidic species, such as lipids, lectins, carbohydrates, vitamins,cofactors, multivalent lactose, multivalent galactose,N-acetyl-galactosamine, N-acetyl-gulucosamine multivalent mannose,multivalent fucose, or aptamers. The ligand can be, for example, alipopolysaccharide, or an activator of p38 MAP kinase.

The targeting group can be any ligand that is capable of targeting aspecific receptor. Examples include, without limitation, folate, GalNAc,galactose, mannose, mannose-6P, apatamers, integrin receptor ligands,chemokine receptor ligands, transferrin, biotin, serotonin receptorligands, PSMA, endothelin, GCPII, somatostatin, LDL, and HDL ligands. Inparticular embodiments, the targeting group is an aptamer. The aptamercan be unmodified or have any combination of modifications disclosedherein.

In one embodiment, pharmaceutical compositions of the present inventionmay include chemical modifications such as, but not limited to,modifications similar to locked nucleic acids.

Representative U.S. patents that teach the preparation of locked nucleicacid (LNA) such as those from Santaris, include, but are not limited to,the following: U.S. Pat. Nos. 6,268,490; 6,670,461; 6,794,499;6,998,484; 7,053,207; 7,084,125; and 7,399,845, each of which is hereinincorporated by reference in its entirety.

Representative U.S. patents that teach the preparation of PNA compoundsinclude, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331;and 5,719,262, each of which is herein incorporated by reference.Further teaching of PNA compounds can be found, for example, in Nielsenet al., Science, 1991, 254, 1497-1500.

Some embodiments featured in the invention include polynucleotides,primary constructs or mmRNA with phosphorothioate backbones andoligonucleosides with other modified backbones, and in particular—CH₂—NH—CH₂—, —CH₂—N(CH₃)—O—CH₂-[known as a methylene (methylimino) orMMI backbone], —CH₂—O—N(CH₃)—CH₂—, —CH₂—N(CH₃)—N(CH₃)—CH₂— and—N(CH₃)—CH₂—CH₂-[wherein the native phosphodiester backbone isrepresented as —O—P(O)₂—O—CH₂—] of the above-referenced U.S. Pat. No.5,489,677, and the amide backbones of the above-referenced U.S. Pat. No.5,602,240. In some embodiments, the polynucletotides featured hereinhave morpholino backbone structures of the above-referenced U.S. Pat.No. 5,034,506.

Modifications at the 2′ position may also aid in delivery. Preferably,modifications at the 2′ position are not located in a polypeptide-codingsequence, i.e., not in a translatable region. Modifications at the 2′position may be located in a 5′UTR, a 3′UTR and/or a tailing region.Modifications at the 2′ position can include one of the following at the2′ position: H (i.e., 2′-deoxy); F; O-, S-, or N-alkyl; O-, S-, orN-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl,alkenyl and alkynyl may be substituted or unsubstituted C₁ to C₁₀ alkylor C₂ to C₁₀ alkenyl and alkynyl. Exemplary suitable modificationsinclude O[(CH₂)_(n)O]_(m)CH₃, O(CH₂)._(n)OCH₃, O(CH₂)_(n)NH₂,O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where nand m are from 1 to about 10. In other embodiments, the polynucleotides,primary constructs or mmRNA include one of the following at the 2′position: C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkaryl,aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃,SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl,aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleavinggroup, a reporter group, an intercalator, a group for improving thepharmacokinetic properties, or a group for improving the pharmacodynamicproperties, and other substituents having similar properties. In someembodiments, the modification includes a 2′-methoxyethoxy(2′-O—CH₂CH₂OCH₃, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martinet al., Helv. Chim. Acta, 1995, 78:486-504) i.e., an alkoxy-alkoxygroup. Another exemplary modification is 2′-dimethylaminooxyethoxy,i.e., a O(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, as described inexamples herein below, and 2′-dimethylaminoethoxyethoxy (also known inthe art as 2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e.,2′-O—CH₂—O—CH₂—N(CH₂)₂, also described in examples herein below. Othermodifications include 2′-methoxy (2′-OCH₃), 2′-aminopropoxy(2′-OCH₂CH₂CH₂NH₂) and 2′-fluoro (2′-F). Similar modifications may alsobe made at other positions, particularly the 3′ position of the sugar onthe 3′ terminal nucleotide or in 2′-5′ linked dsRNAs and the 5′ positionof 5′ terminal nucleotide. polynucleotides of the invention may alsohave sugar mimetics such as cyclobutyl moieties in place of thepentofuranosyl sugar. Representative U.S. patents that teach thepreparation of such modified sugar structures include, but are notlimited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044;5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811;5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873;5,646,265; 5,658,873; 5,670,633; and 5,700,920 and each of which isherein incorporated by reference.

In still other embodiments, the polynucleotide, primary construct, ormmRNA is covalently conjugated to a cell penetrating polypeptide. Thecell-penetrating peptide may also include a signal sequence. Theconjugates of the invention can be designed to have increased stability;increased cell transfection; and/or altered the biodistribution (e.g.,targeted to specific tissues or cell types).

Self-Assembled Nucleic Acid Nanoparticles

Self-assembled nanoparticles have a well-defined size which may beprecisely controlled as the nucleic acid strands may be easilyreprogrammable. For example, the optimal particle size for acancer-targeting nanodelivery carrier is 20-100 nm as a diameter greaterthan 20 nm avoids renal clearance and enhances delivery to certaintumors through enhanced permeability and retention effect. Usingself-assembled nucleic acid nanoparticles a single uniform population insize and shape having a precisely controlled spatial orientation anddensity of cancer-targeting ligands for enhanced delivery. As anon-limiting example, oligonucleotide nanoparticles are prepared usingprogrammable self-assembly of short DNA fragments and therapeuticsiRNAs. These nanoparticles are molecularly identical with controllableparticle size and target ligand location and density. The DNA fragmentsand siRNAs self-assembled into a one-step reaction to generate DNA/siRNAtetrahedral nanoparticles for targeted in vivo delivery. (Lee et al.,Nature Nanotechnology 2012 7:389-393).

Excipients

Pharmaceutical formulations may additionally comprise a pharmaceuticallyacceptable excipient, which, as used herein, includes any and allsolvents, dispersion media, diluents, or other liquid vehicles,dispersion or suspension aids, surface active agents, isotonic agents,thickening or emulsifying agents, preservatives, solid binders,lubricants and the like, as suited to the particular dosage formdesired. Remington's The Science and Practice of Pharmacy, 21^(st)Edition, A. R. Gennaro (Lippincott, Williams & Wilkins, Baltimore, Md.,2006; incorporated herein by reference) discloses various excipientsused in formulating pharmaceutical compositions and known techniques forthe preparation thereof. Except insofar as any conventional excipientmedium is incompatible with a substance or its derivatives, such as byproducing any undesirable biological effect or otherwise interacting ina deleterious manner with any other component(s) of the pharmaceuticalcomposition, its use is contemplated to be within the scope of thisinvention.

In some embodiments, a pharmaceutically acceptable excipient is at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%pure. In some embodiments, an excipient is approved for use in humansand for veterinary use. In some embodiments, an excipient is approved byUnited States Food and Drug Administration. In some embodiments, anexcipient is pharmaceutical grade. In some embodiments, an excipientmeets the standards of the United States Pharmacopoeia (USP), theEuropean Pharmacopoeia (EP), the British Pharmacopoeia, and/or theInternational Pharmacopoeia.

Pharmaceutically acceptable excipients used in the manufacture ofpharmaceutical compositions include, but are not limited to, inertdiluents, dispersing and/or granulating agents, surface active agentsand/or emulsifiers, disintegrating agents, binding agents,preservatives, buffering agents, lubricating agents, and/or oils. Suchexcipients may optionally be included in pharmaceutical compositions.

Exemplary diluents include, but are not limited to, calcium carbonate,sodium carbonate, calcium phosphate, dicalcium phosphate, calciumsulfate, calcium hydrogen phosphate, sodium phosphate lactose, sucrose,cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol,inositol, sodium chloride, dry starch, cornstarch, powdered sugar, etc.,and/or combinations thereof.

Exemplary granulating and/or dispersing agents include, but are notlimited to, potato starch, corn starch, tapioca starch, sodium starchglycolate, clays, alginic acid, guar gum, citrus pulp, agar, bentonite,cellulose and wood products, natural sponge, cation-exchange resins,calcium carbonate, silicates, sodium carbonate, cross-linkedpoly(vinyl-pyrrolidone) (crospovidone), sodium carboxymethyl starch(sodium starch glycolate), carboxymethyl cellulose, cross-linked sodiumcarboxymethyl cellulose (croscarmellose), methylcellulose,pregelatinized starch (starch 1500), microcrystalline starch, waterinsoluble starch, calcium carboxymethyl cellulose, magnesium aluminumsilicate (VEEGUM®), sodium lauryl sulfate, quaternary ammoniumcompounds, etc., and/or combinations thereof.

Exemplary surface active agents and/or emulsifiers include, but are notlimited to, natural emulsifiers (e.g. acacia, agar, alginic acid, sodiumalginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin,egg yolk, casein, wool fat, cholesterol, wax, and lecithin), colloidalclays (e.g. bentonite [aluminum silicate] and VEEGUM® [magnesiumaluminum silicate]), long chain amino acid derivatives, high molecularweight alcohols (e.g. stearyl alcohol, cetyl alcohol, oleyl alcohol,triacetin monostearate, ethylene glycol distearate, glycerylmonostearate, and propylene glycol monostearate, polyvinyl alcohol),carbomers (e.g. carboxy polymethylene, polyacrylic acid, acrylic acidpolymer, and carboxyvinyl polymer), carrageenan, cellulosic derivatives(e.g. carboxymethylcellulose sodium, powdered cellulose, hydroxymethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose,methylcellulose), sorbitan fatty acid esters (e.g. polyoxyethylenesorbitan monolaurate [TWEEN® 20], polyoxyethylene sorbitan [TWEENn® 60],polyoxyethylene sorbitan monooleate [TWEEN® 80], sorbitan monopalmitate[SPAN® 40], sorbitan monostearate [Span® 60], sorbitan tristearate [Span6], glyceryl monooleate, sorbitan monooleate [SPAN® 80]),polyoxyethylene esters (e.g. polyoxyethylene monostearate [MYRJ® 45],polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil,polyoxymethylene stearate, and SOLUTOL), sucrose fatty acid esters,polyethylene glycol fatty acid esters (e.g. CREMOPHOR®), polyoxyethyleneethers, (e.g. polyoxyethylene lauryl ether [BRIJ®30]),poly(vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamineoleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyllaurate, sodium lauryl sulfate, PLUORINC®F 68, POLOXAMER®188,cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride,docusate sodium, etc. and/or combinations thereof.

Exemplary binding agents include, but are not limited to, starch (e.g.cornstarch and starch paste); gelatin; sugars (e.g. sucrose, glucose,dextrose, dextrin, molasses, lactose, lactitol, mannitol,); natural andsynthetic gums (e.g. acacia, sodium alginate, extract of Irish moss,panwar gum, ghatti gum, mucilage of isapol husks,carboxymethylcellulose, methylcellulose, ethylcellulose,hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropylmethylcellulose, microcrystalline cellulose, cellulose acetate,poly(vinyl-pyrrolidone), magnesium aluminum silicate)(Veegum®, and larcharabogalactan); alginates; polyethylene oxide; polyethylene glycol;inorganic calcium salts; silicic acid; polymethacrylates; waxes; water;alcohol; etc.; and combinations thereof.

Exemplary preservatives may include, but are not limited to,antioxidants, chelating agents, antimicrobial preservatives, antifungalpreservatives, alcohol preservatives, acidic preservatives, and/or otherpreservatives. Exemplary antioxidants include, but are not limited to,alpha tocopherol, ascorbic acid, acorbyl palmitate, butylatedhydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassiummetabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodiumbisulfite, sodium metabisulfite, and/or sodium sulfite. Exemplarychelating agents include ethylenediaminetetraacetic acid (EDTA), citricacid monohydrate, disodium edetate, dipotassium edetate, edetic acid,fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaricacid, and/or trisodium edetate. Exemplary antimicrobial preservativesinclude, but are not limited to, benzalkonium chloride, benzethoniumchloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride,chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethylalcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol,phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and/orthimerosal. Exemplary antifungal preservatives include, but are notlimited to, butyl paraben, methyl paraben, ethyl paraben, propylparaben, benzoic acid, hydroxybenzoic acid, potassium benzoate,potassium sorbate, sodium benzoate, sodium propionate, and/or sorbicacid. Exemplary alcohol preservatives include, but are not limited to,ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenol,chlorobutanol, hydroxybenzoate, and/or phenylethyl alcohol. Exemplaryacidic preservatives include, but are not limited to, vitamin A, vitaminC, vitamin E, beta-carotene, citric acid, acetic acid, dehydroaceticacid, ascorbic acid, sorbic acid, and/or phytic acid. Otherpreservatives include, but are not limited to, tocopherol, tocopherolacetate, deteroxime mesylate, cetrimide, butylated hydroxyanisol (BHA),butylated hydroxytoluened (BHT), ethylenediamine, sodium lauryl sulfate(SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodiummetabisulfite, potassium sulfite, potassium metabisulfite, GLYDANTPLUS®, PHENONIP®, methylparaben, GERMALL® 115, GERMABEN®II, NEOLONE™KATHON™, and/or EUXYL®.

Exemplary buffering agents include, but are not limited to, citratebuffer solutions, acetate buffer solutions, phosphate buffer solutions,ammonium chloride, calcium carbonate, calcium chloride, calcium citrate,calcium glubionate, calcium gluceptate, calcium gluconate, D-gluconicacid, calcium glycerophosphate, calcium lactate, propanoic acid, calciumlevulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid,tribasic calcium phosphate, calcium hydroxide phosphate, potassiumacetate, potassium chloride, potassium gluconate, potassium mixtures,dibasic potassium phosphate, monobasic potassium phosphate, potassiumphosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride,sodium citrate, sodium lactate, dibasic sodium phosphate, monobasicsodium phosphate, sodium phosphate mixtures, tromethamine, magnesiumhydroxide, aluminum hydroxide, alginic acid, pyrogen-free water,isotonic saline, Ringer's solution, ethyl alcohol, etc., and/orcombinations thereof.

Exemplary lubricating agents include, but are not limited to, magnesiumstearate, calcium stearate, stearic acid, silica, talc, malt, glycerylbehanate, hydrogenated vegetable oils, polyethylene glycol, sodiumbenzoate, sodium acetate, sodium chloride, leucine, magnesium laurylsulfate, sodium lauryl sulfate, etc., and combinations thereof.

Exemplary oils include, but are not limited to, almond, apricot kernel,avocado, babassu, bergamot, black current seed, borage, cade, camomile,canola, caraway, carnauba, castor, cinnamon, cocoa butter, coconut, codliver, coffee, corn, cotton seed, emu, eucalyptus, evening primrose,fish, flaxseed, geraniol, gourd, grape seed, hazel nut, hyssop,isopropyl myristate, jojoba, kukui nut, lavandin, lavender, lemon,litsea cubeba, macademia nut, mallow, mango seed, meadowfoam seed, mink,nutmeg, olive, orange, orange roughy, palm, palm kernel, peach kernel,peanut, poppy seed, pumpkin seed, rapeseed, rice bran, rosemary,safflower, sandalwood, sasquana, savoury, sea buckthorn, sesame, sheabutter, silicone, soybean, sunflower, tea tree, thistle, tsubaki,vetiver, walnut, and wheat germ oils. Exemplary oils include, but arenot limited to, butyl stearate, caprylic triglyceride, caprictriglyceride, cyclomethicone, diethyl sebacate, dimethicone 360,isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol,silicone oil, and/or combinations thereof.

Excipients such as cocoa butter and suppository waxes, coloring agents,coating agents, sweetening, flavoring, and/or perfuming agents can bepresent in the composition, according to the judgment of the formulator.

Delivery

The present disclosure encompasses the delivery of polynucleotides,primary constructs or mmRNA for any of therapeutic, pharmaceutical,diagnostic or imaging by any appropriate route taking into considerationlikely advances in the sciences of drug delivery. Delivery may be nakedor formulated.

Naked Delivery

The polynucleotides, primary constructs or mmRNA of the presentinvention may be delivered to a cell naked. As used herein in, “naked”refers to delivering polynucleotides, primary constructs or mmRNA freefrom agents which promote transfection. For example, thepolynucleotides, primary constructs or mmRNA delivered to the cell maycontain no modifications. The naked polynucleotides, primary constructsor mmRNA may be delivered to the cell using routes of administrationknown in the art and described herein.

Formulated Delivery

The polynucleotides, primary constructs or mmRNA of the presentinvention may be formulated, using the methods described herein. Theformulations may contain polynucleotides, primary constructs or mmRNAwhich may be modified and/or unmodified. The formulations may furtherinclude, but are not limited to, cell penetration agents, apharmaceutically acceptable carrier, a delivery agent, a bioerodible orbiocompatible polymer, a solvent, and a sustained-release deliverydepot. The formulated polynucleotides, primary constructs or mmRNA maybe delivered to the cell using routes of administration known in the artand described herein.

The compositions may also be formulated for direct delivery to an organor tissue in any of several ways in the art including, but not limitedto, direct soaking or bathing, via a catheter, by gels, powder,ointments, creams, gels, lotions, and/or drops, by using substrates suchas fabric or biodegradable materials coated or impregnated with thecompositions, and the like.

Administration

The polynucleotides, primary constructs or mmRNA of the presentinvention may be administered by any route which results in atherapeutically effective outcome. These include, but are not limited toenteral, gastroenteral, epidural, oral, transdermal, epidural(peridural), intracerebral (into the cerebrum), intracerebroventricular(into the cerebral ventricles), epicutaneous (application onto theskin), intradermal, (into the skin itself), subcutaneous (under theskin), nasal administration (through the nose), intravenous (into avein), intraarterial (into an artery), intramuscular (into a muscle),intracardiac (into the heart), intraosseous infusion (into the bonemarrow), intrathecal (into the spinal canal), intraperitoneal, (infusionor injection into the peritoneum), intravesical infusion, intravitreal,(through the eye), intracavernous injection, (into the base of thepenis), intravaginal administration, intrauterine, extra-amnioticadministration, transdermal (diffusion through the intact skin forsystemic distribution), transmucosal (diffusion through a mucousmembrane), insufflation (snorting), sublingual, sublabial, enema, eyedrops (onto the conjunctiva), or in ear drops. In specific embodiments,compositions may be administered in a way which allows them cross theblood-brain barrier, vascular barrier, or other epithelial barrier.Non-limiting routes of administration for the polynucleotides, primaryconstructs or mmRNA of the present invention are described below.

Parenteral and Injectible Administration

Liquid dosage forms for oral and parenteral administration include, butare not limited to, pharmaceutically acceptable emulsions,microemulsions, solutions, suspensions, syrups, and/or elixirs. Inaddition to active ingredients, liquid dosage forms may comprise inertdiluents commonly used in the art such as, for example, water or othersolvents, solubilizing agents and emulsifiers such as ethyl alcohol,isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol,benzyl benzoate, propylene glycol, 1,3-butylene glycol,dimethylformamide, oils (in particular, cottonseed, groundnut, corn,germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfurylalcohol, polyethylene glycols and fatty acid esters of sorbitan, andmixtures thereof. Besides inert diluents, oral compositions can includeadjuvants such as wetting agents, emulsifying and suspending agents,sweetening, flavoring, and/or perfuming agents. In certain embodimentsfor parenteral administration, compositions are mixed with solubilizingagents such as CREMOPHOR, alcohols, oils, modified oils, glycols,polysorbates, cyclodextrins, polymers, and/or combinations thereof.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing agents, wetting agents, and/or suspendingagents. Sterile injectable preparations may be sterile injectablesolutions, suspensions, and/or emulsions in nontoxic parenterallyacceptable diluents and/or solvents, for example, as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that may beemployed are water, Ringer's solution, U.S.P., and isotonic sodiumchloride solution. Sterile, fixed oils are conventionally employed as asolvent or suspending medium. For this purpose any bland fixed oil canbe employed including synthetic mono- or diglycerides. Fatty acids suchas oleic acid can be used in the preparation of injectables.

Injectable formulations can be sterilized, for example, by filtrationthrough a bacterial-retaining filter, and/or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium prior to use.

In order to prolong the effect of an active ingredient, it is oftendesirable to slow the absorption of the active ingredient fromsubcutaneous or intramuscular injection. This may be accomplished by theuse of a liquid suspension of crystalline or amorphous material withpoor water solubility. The rate of absorption of the drug then dependsupon its rate of dissolution which, in turn, may depend upon crystalsize and crystalline form. Alternatively, delayed absorption of aparenterally administered drug form is accomplished by dissolving orsuspending the drug in an oil vehicle. Injectable depot forms are madeby forming microencapsule matrices of the drug in biodegradable polymerssuch as polylactide-polyglycolide. Depending upon the ratio of drug topolymer and the nature of the particular polymer employed, the rate ofdrug release can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are prepared by entrapping the drug in liposomes ormicroemulsions which are compatible with body tissues.

Rectal and Vaginal Administration

Compositions for rectal or vaginal administration are typicallysuppositories which can be prepared by mixing compositions with suitablenon-irritating excipients such as cocoa butter, polyethylene glycol or asuppository wax which are solid at ambient temperature but liquid atbody temperature and therefore melt in the rectum or vaginal cavity andrelease the active ingredient.

Oral Administration

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, an activeingredient is mixed with at least one inert, pharmaceutically acceptableexcipient such as sodium citrate or dicalcium phosphate and/or fillersor extenders (e.g. starches, lactose, sucrose, glucose, mannitol, andsilicic acid), binders (e.g. carboxymethylcellulose, alginates, gelatin,polyvinylpyrrolidinone, sucrose, and acacia), humectants (e.g.glycerol), disintegrating agents (e.g. agar, calcium carbonate, potatoor tapioca starch, alginic acid, certain silicates, and sodiumcarbonate), solution retarding agents (e.g. paraffin), absorptionaccelerators (e.g. quaternary ammonium compounds), wetting agents (e.g.cetyl alcohol and glycerol monostearate), absorbents (e.g. kaolin andbentonite clay), and lubricants (e.g. talc, calcium stearate, magnesiumstearate, solid polyethylene glycols, sodium lauryl sulfate), andmixtures thereof. In the case of capsules, tablets and pills, the dosageform may comprise buffering agents.

Topical or Transdermal Administration

As described herein, compositions containing the polynucleotides,primary constructs or mmRNA of the invention may be formulated foradministration topically. The skin may be an ideal target site fordelivery as it is readily accessible. Gene expression may be restrictednot only to the skin, potentially avoiding nonspecific toxicity, butalso to specific layers and cell types within the skin.

The site of cutaneous expression of the delivered compositions willdepend on the route of nucleic acid delivery. Three routes are commonlyconsidered to deliver polynucleotides, primary constructs or mmRNA tothe skin: (i) topical application (e.g. for local/regional treatmentand/or cosmetic applications); (ii) intradermal injection (e.g. forlocal/regional treatment and/or cosmetic applications); and (iii)systemic delivery (e.g. for treatment of dermatologic diseases thataffect both cutaneous and extracutaneous regions). polynucleotides,primary constructs or mmRNA can be delivered to the skin by severaldifferent approaches known in the art. Most topical delivery approacheshave been shown to work for delivery of DNA, such as but not limited to,topical application of non-cationic liposome-DNA complex, cationicliposome-DNA complex, particle-mediated (gene gun), puncture-mediatedgene transfections, and viral delivery approaches. After delivery of thenucleic acid, gene products have been detected in a number of differentskin cell types, including, but not limited to, basal keratinocytes,sebaceous gland cells, dermal fibroblasts and dermal macrophages.

In one embodiment, the invention provides for a variety of dressings(e.g., wound dressings) or bandages (e.g., adhesive bandages) forconveniently and/or effectively carrying out methods of the presentinvention. Typically dressing or bandages may comprise sufficientamounts of pharmaceutical compositions and/or polynucleotides, primaryconstructs or mmRNA described herein to allow a user to perform multipletreatments of a subject(s).

In one embodiment, the invention provides for the polynucleotides,primary constructs or mmRNA compositions to be delivered in more thanone injection.

In one embodiment, before topical and/or transdermal administration atleast one area of tissue, such as skin, may be subjected to a deviceand/or solution which may increase permeability. In one embodiment, thetissue may be subjected to an abrasion device to increase thepermeability of the skin (see U.S. Patent Publication No. 20080275468,herein incorporated by reference in its entirety). In anotherembodiment, the tissue may be subjected to an ultrasound enhancementdevice. An ultrasound enhancement device may include, but is not limitedto, the devices described in U.S. Publication No. 20040236268 and U.S.Pat. Nos. 6,491,657 and 6,234,990; each of which is herein incorporatedby reference in their entireties. Methods of enhancing the permeabilityof tissue are described in U.S. Publication Nos. 20040171980 and20040236268 and U.S. Pat. No. 6,190,315; each of whish are hereinincorporated by reference in their entireties.

In one embodiment, a device may be used to increase permeability oftissue before delivering formulations of the polynucleotides, primaryconstructs and mmRNA described herein. The permeability of skin may bemeasured by methods known in the art and/or described in U.S. Pat. No.6,190,315, herein incorporated by reference in its entirety. As anon-limiting example, a modified mRNA formulation may be delivered bythe drug delivery methods described in U.S. Pat. No. 6,190,315, hereinincorporated by reference in its entirety.

In another non-limiting example tissue may be treated with a eutecticmixture of local anesthetics (EMLA) cream before, during and/or afterthe tissue may be subjected to a device which may increase permeability.Katz et al. (Anesth Analg (2004); 98:371-76; herein incorporated byreference in its entirety) showed that using the EMLA cream incombination with a low energy, an onset of superficial cutaneousanalgesia was seen as fast as 5 minutes after a pretreatment with a lowenergy ultrasound.

In one embodiment, enhancers may be applied to the tissue before,during, and/or after the tissue has been treated to increasepermeability. Enhancers include, but are not limited to, transportenhancers, physical enhancers, and cavitation enhancers. Non-limitingexamples of enhancers are described in U.S. Pat. No. 6,190,315, hereinincorporated by reference in its entirety.

In one embodiment, a device may be used to increase permeability oftissue before delivering formulations of polynucleotides, primaryconstructs and/or mmRNA described herein, which may further contain asubstance that invokes an immune response. In another non-limitingexample, a formulation containing a substance to invoke an immuneresponse may be delivered by the methods described in U.S. PublicationNos. 20040171980 and 20040236268; each of which is herein incorporatedby reference in their entirety.

Dosage forms for topical and/or transdermal administration of acomposition may include ointments, pastes, creams, lotions, gels,powders, solutions, sprays, inhalants and/or patches. Generally, anactive ingredient is admixed under sterile conditions with apharmaceutically acceptable excipient and/or any needed preservativesand/or buffers as may be required.

Additionally, the present invention contemplates the use of transdermalpatches, which often have the added advantage of providing controlleddelivery of a compound to the body. Such dosage forms may be prepared,for example, by dissolving and/or dispensing the compound in the propermedium. Alternatively or additionally, rate may be controlled by eitherproviding a rate controlling membrane and/or by dispersing the compoundin a polymer matrix and/or gel.

Formulations suitable for topical administration include, but are notlimited to, liquid and/or semi liquid preparations such as liniments,lotions, oil in water and/or water in oil emulsions such as creams,ointments and/or pastes, and/or solutions and/or suspensions.Topically-administrable formulations may, for example, comprise fromabout 0.1% to about 10% (w/w) active ingredient, although theconcentration of active ingredient may be as high as the solubilitylimit of the active ingredient in the solvent. Formulations for topicaladministration may further comprise one or more of the additionalingredients described herein.

Depot Administration

As described herein, in some embodiments, the composition is formulatedin depots for extended release. Generally, a specific organ or tissue (a“target tissue”) is targeted for administration.

In some aspects of the invention, the polynucleotides, primaryconstructs or mmRNA are spatially retained within or proximal to atarget tissue. Provided are method of providing a composition to atarget tissue of a mammalian subject by contacting the target tissue(which contains one or more target cells) with the composition underconditions such that the composition, in particular the nucleic acidcomponent(s) of the composition, is substantially retained in the targettissue, meaning that at least 10, 20, 30, 40, 50, 60, 70, 80, 85, 90,95, 96, 97, 98, 99, 99.9, 99.99 or greater than 99.99% of thecomposition is retained in the target tissue. Advantageously, retentionis determined by measuring the amount of the nucleic acid present in thecomposition that enters one or more target cells. For example, at least1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99, 99.9,99.99 or greater than 99.99% of the nucleic acids administered to thesubject are present intracellularly at a period of time followingadministration. For example, intramuscular injection to a mammaliansubject is performed using an aqueous composition containing aribonucleic acid and a transfection reagent, and retention of thecomposition is determined by measuring the amount of the ribonucleicacid present in the muscle cells.

Aspects of the invention are directed to methods of providing acomposition to a target tissue of a mammalian subject, by contacting thetarget tissue (containing one or more target cells) with the compositionunder conditions such that the composition is substantially retained inthe target tissue. The composition contains an effective amount of apolynucleotide, primary construct or mmRNA such that the polypeptide ofinterest is produced in at least one target cell. The compositionsgenerally contain a cell penetration agent, although “naked” nucleicacid (such as nucleic acids without a cell penetration agent or otheragent) is also contemplated, and a pharmaceutically acceptable carrier.

In some circumstances, the amount of a protein produced by cells in atissue is desirably increased. Preferably, this increase in proteinproduction is spatially restricted to cells within the target tissue.Thus, provided are methods of increasing production of a protein ofinterest in a tissue of a mammalian subject. A composition is providedthat contains polynucleotides, primary constructs or mmRNA characterizedin that a unit quantity of composition has been determined to producethe polypeptide of interest in a substantial percentage of cellscontained within a predetermined volume of the target tissue.

In some embodiments, the composition includes a plurality of differentpolynucleotides, primary constructs or mmRNA, where one or more than oneof the polynucleotides, primary constructs or mmRNA encodes apolypeptide of interest. Optionally, the composition also contains acell penetration agent to assist in the intracellular delivery of thecomposition. A determination is made of the dose of the compositionrequired to produce the polypeptide of interest in a substantialpercentage of cells contained within the predetermined volume of thetarget tissue (generally, without inducing significant production of thepolypeptide of interest in tissue adjacent to the predetermined volume,or distally to the target tissue). Subsequent to this determination, thedetermined dose is introduced directly into the tissue of the mammaliansubject.

In one embodiment, the invention provides for the polynucleotides,primary constructs or mmRNA to be delivered in more than one injectionor by split dose injections.

In one embodiment, the invention may be retained near target tissueusing a small disposable drug reservoir or patch pump. Non-limitingexamples of patch pumps include those manufactured and/or sold by BD®(Franklin Lakes, N.J.), Insulet Corporation (Bedford, Mass.), SteadyMedTherapeutics (San Francisco, Calif.), Medtronic (Minneapolis, Minn.),UniLife (York, Pa.), Valeritas (Bridgewater, N.J.), and SpringLeafTherapeutics (Boston, Mass.).

Pulmonary Administration

A pharmaceutical composition may be prepared, packaged, and/or sold in aformulation suitable for pulmonary administration via the buccal cavity.Such a formulation may comprise dry particles which comprise the activeingredient and which have a diameter in the range from about 0.5 nm toabout 7 nm or from about 1 nm to about 6 nm. Such compositions aresuitably in the form of dry powders for administration using a devicecomprising a dry powder reservoir to which a stream of propellant may bedirected to disperse the powder and/or using a self propellingsolvent/powder dispensing container such as a device comprising theactive ingredient dissolved and/or suspended in a low-boiling propellantin a sealed container. Such powders comprise particles wherein at least98% of the particles by weight have a diameter greater than 0.5 nm andat least 95% of the particles by number have a diameter less than 7 nm.Alternatively, at least 95% of the particles by weight have a diametergreater than 1 nm and at least 90% of the particles by number have adiameter less than 6 nm. Dry powder compositions may include a solidfine powder diluent such as sugar and are conveniently provided in aunit dose form.

Low boiling propellants generally include liquid propellants having aboiling point of below 65° F. at atmospheric pressure. Generally thepropellant may constitute 50% to 99.9% (w/w) of the composition, andactive ingredient may constitute 0.1% to 20% (w/w) of the composition. Apropellant may further comprise additional ingredients such as a liquidnon-ionic and/or solid anionic surfactant and/or a solid diluent (whichmay have a particle size of the same order as particles comprising theactive ingredient).

Pharmaceutical compositions formulated for pulmonary delivery mayprovide an active ingredient in the form of droplets of a solutionand/or suspension. Such formulations may be prepared, packaged, and/orsold as aqueous and/or dilute alcoholic solutions and/or suspensions,optionally sterile, comprising active ingredient, and may convenientlybe administered using any nebulization and/or atomization device. Suchformulations may further comprise one or more additional ingredientsincluding, but not limited to, a flavoring agent such as saccharinsodium, a volatile oil, a buffering agent, a surface active agent,and/or a preservative such as methylhydroxybenzoate. Droplets providedby this route of administration may have an average diameter in therange from about 0.1 nm to about 200 nm.

Intranasal, Nasal and Buccal Administration

Formulations described herein as being useful for pulmonary delivery areuseful for intranasal delivery of a pharmaceutical composition. Anotherformulation suitable for intranasal administration is a coarse powdercomprising the active ingredient and having an average particle fromabout 0.2 μm to 500 μm. Such a formulation is administered in the mannerin which snuff is taken, i.e. by rapid inhalation through the nasalpassage from a container of the powder held close to the nose.

Formulations suitable for nasal administration may, for example,comprise from about as little as 0.1% (w/w) and as much as 100% (w/w) ofactive ingredient, and may comprise one or more of the additionalingredients described herein. A pharmaceutical composition may beprepared, packaged, and/or sold in a formulation suitable for buccaladministration. Such formulations may, for example, be in the form oftablets and/or lozenges made using conventional methods, and may, forexample, 0.1% to 20% (w/w) active ingredient, the balance comprising anorally dissolvable and/or degradable composition and, optionally, one ormore of the additional ingredients described herein. Alternately,formulations suitable for buccal administration may comprise a powderand/or an aerosolized and/or atomized solution and/or suspensioncomprising active ingredient. Such powdered, aerosolized, and/oraerosolized formulations, when dispersed, may have an average particleand/or droplet size in the range from about 0.1 nm to about 200 nm, andmay further comprise one or more of any additional ingredients describedherein.

Ophthalmic Administration

A pharmaceutical composition may be prepared, packaged, and/or sold in aformulation suitable for ophthalmic administration. Such formulationsmay, for example, be in the form of eye drops including, for example, a0.1/1.0% (w/w) solution and/or suspension of the active ingredient in anaqueous or oily liquid excipient. Such drops may further comprisebuffering agents, salts, and/or one or more other of any additionalingredients described herein. Other ophthalmically-administrableformulations which are useful include those which comprise the activeingredient in microcrystalline form and/or in a liposomal preparation.Ear drops and/or eye drops are contemplated as being within the scope ofthis invention.

Payload Administration: Detectable Agents and Therapeutic Agents

The polynucleotides, primary constructs or mmRNA described herein can beused in a number of different scenarios in which delivery of a substance(the “payload”) to a biological target is desired, for example deliveryof detectable substances for detection of the target, or delivery of atherapeutic agent. Detection methods can include, but are not limitedto, both imaging in vitro and in vivo imaging methods, e.g.,immunohistochemistry, bioluminescence imaging (BLI), Magnetic ResonanceImaging (MM), positron emission tomography (PET), electron microscopy,X-ray computed tomography, Raman imaging, optical coherence tomography,absorption imaging, thermal imaging, fluorescence reflectance imaging,fluorescence microscopy, fluorescence molecular tomographic imaging,nuclear magnetic resonance imaging, X-ray imaging, ultrasound imaging,photoacoustic imaging, lab assays, or in any situation wheretagging/staining/imaging is required.

The polynucleotides, primary constructs or mmRNA can be designed toinclude both a linker and a payload in any useful orientation. Forexample, a linker having two ends is used to attach one end to thepayload and the other end to the nucleobase, such as at the C-7 or C-8positions of the deaza-adenosine or deaza-guanosine or to the N-3 or C-5positions of cytosine or uracil. The polynucleotide of the invention caninclude more than one payload (e.g., a label and a transcriptioninhibitor), as well as a cleavable linker. In one embodiment, themodified nucleotide is a modified 7-deaza-adenosine triphosphate, whereone end of a cleavable linker is attached to the C7 position of7-deaza-adenine, the other end of the linker is attached to an inhibitor(e.g., to the C5 position of the nucleobase on a cytidine), and a label(e.g., Cy5) is attached to the center of the linker (see, e.g., compound1 of A*pCp C5 Parg Capless in FIG. 5 and columns 9 and 10 of U.S. Pat.No. 7,994,304, incorporated herein by reference). Upon incorporation ofthe modified 7-deaza-adenosine triphosphate to an encoding region, theresulting polynucleotide will have a cleavable linker attached to alabel and an inhibitor (e.g., a polymerase inhibitor). Upon cleavage ofthe linker (e.g., with reductive conditions to reduce a linker having acleavable disulfide moiety), the label and inhibitor are released.Additional linkers and payloads (e.g., therapeutic agents, detectablelabels, and cell penetrating payloads) are described herein.

For example, the polynucleotides, primary constructs or mmRNA describedherein can be used in induced pluripotent stem cells (iPS cells), whichcan directly track cells that are transfected compared to total cells inthe cluster. In another example, a drug that may be attached to thepolynucleotides, primary constructs or mmRNA via a linker and may befluorescently labeled can be used to track the drug in vivo, e.g.intracellularly. Other examples include, but are not limited to, the useof a polynucleotide, primary construct or mmRNA in reversible drugdelivery into cells.

The polynucleotides, primary constructs or mmRNA described herein can beused in intracellular targeting of a payload, e.g., detectable ortherapeutic agent, to specific organelle. Exemplary intracellulartargets can include, but are not limited to, the nuclear localizationfor advanced mRNA processing, or a nuclear localization sequence (NLS)linked to the mRNA containing an inhibitor.

In addition, the polynucleotides, primary constructs or mmRNA describedherein can be used to deliver therapeutic agents to cells or tissues,e.g., in living animals. For example, the polynucleotides, primaryconstructs or mmRNA attached to the therapeutic agent through a linkercan facilitate member permeation allowing the therapeutic agent totravel into a cell to reach an intracellular target.

In some embodiments, the payload may be a therapeutic agent such as acytotoxin, radioactive ion, chemotherapeutic, or other therapeuticagent. A cytotoxin or cytotoxic agent includes any agent that may bedetrimental to cells. Examples include, but are not limited to, taxol,cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin,etoposide, teniposide, vincristine, vinblastine, colchicine,doxorubicin, daunorubicin, dihydroxyanthracinedione, mitoxantrone,mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids,procaine, tetracaine, lidocaine, propranolol, puromycin, maytansinoids,e.g., maytansinol (see U.S. Pat. No. 5,208,020 incorporated herein inits entirety), rachelmycin (CC-1065, see U.S. Pat. Nos. 5,475,092,5,585,499, and 5,846,545, all of which are incorporated herein byreference), and analogs or homologs thereof. Radioactive ions include,but are not limited to iodine (e.g., iodine 125 or iodine 131),strontium 89, phosphorous, palladium, cesium, iridium, phosphate,cobalt, yttrium 90, samarium 153, and praseodymium. Other therapeuticagents include, but are not limited to, antimetabolites (e.g.,methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine,5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine,thiotepa chlorambucil, rachelmycin (CC-1065), melphalan, carmustine(BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol,streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine, vinblastine, taxol and maytansinoids).

In some embodiments, the payload may be a detectable agent, such asvarious organic small molecules, inorganic compounds, nanoparticles,enzymes or enzyme substrates, fluorescent materials, luminescentmaterials (e.g., luminol), bioluminescent materials (e.g., luciferase,luciferin, and aequorin), chemiluminescent materials, radioactivematerials (e.g., 18F, ⁶⁷Ga, ^(81m)Kr, ⁸²Rb, ¹¹¹In, ¹²³I, ¹³³Xe, ²⁰¹Tl,¹²⁵I, ³⁵S, ¹⁴C, ³H, or ^(99m)Tc (e.g., as pertechnetate(technetate(VII), TcO₄ ⁻)), and contrast agents (e.g., gold (e.g., goldnanoparticles), gadolinium (e.g., chelated Gd), iron oxides (e.g.,superparamagnetic iron oxide (SPIO), monocrystalline iron oxidenanoparticles (MIONs), and ultrasmall superparamagnetic iron oxide(USPIO)), manganese chelates (e.g., Mn-DPDP), barium sulfate, iodinatedcontrast media (iohexol), microbubbles, or perfluorocarbons). Suchoptically-detectable labels include for example, without limitation,4-acetamido-4′-isothiocyanatostilbene-2,2′disulfonic acid; acridine andderivatives (e.g., acridine and acridine isothiocyanate);5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS);4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate;N-(4-anilino-1-naphthyl)maleimide; anthranilamide; BODIPY; BrilliantYellow; coumarin and derivatives (e.g., coumarin,7-amino-4-methylcoumarin (AMC, Coumarin 120), and7-amino-4-trifluoromethylcoumarin (Coumarin 151)); cyanine dyes;cyanosine; 4′,6-diaminidino-2-phenylindole (DAPI); 5′5″-dibromopyrogallol-sulfonaphthalein (Bromopyrogallol Red);7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarin;diethylenetriamine pentaacetate;4,4′-diisothiocyanatodihydro-stilbene-2,2′-disulfonic acid;4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid;5-[dimethylamino]-naphthalene-1-sulfonyl chloride (DNS, dansylchloride);4-dimethylaminophenylazophenyl-4′-isothiocyanate (DABITC); eosin andderivatives (e.g., eosin and eosin isothiocyanate); erythrosin andderivatives (e.g., erythrosin B and erythrosin isothiocyanate);ethidium; fluorescein and derivatives (e.g., 5-carboxyfluorescein (FAM),dichlorotriazin-2-yl)aminofluorescein (DTAF),2′,7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein, fluorescein,fluorescein isothiocyanate, X-rhodamine-5-(and-6)-isothiocyanate (QFITCor XRITC), and fluorescamine);2-[2-[3-[[1,3-dihydro-1,1-dimethyl-3-(3-sulfopropyl)-2H-benz[e]indol-2-ylidene]ethylidene]-2-[4-(ethoxycarbonyl)-1-piperazinyl]-1-cyclopenten-1-yl]ethenyl]-1,1-dimethyl-3-(3-sulforpropyl)-1H-benz[e]indoliumhydroxide, inner salt, compound with n,n-diethylethanamine(1:1) (IR144);5-chloro-2-[2-[3-[(5-chloro-3-ethyl-2(3H)-benzothiazol-ylidene)ethylidene]-2-(diphenylamino)-1-cyclopenten-1-yl]ethenyl]-3-ethylbenzothiazolium perchlorate (IR140); Malachite Green isothiocyanate;4-methylumbelliferone orthocresolphthalein; nitrotyrosine;pararosaniline; Phenol Red; B-phycoerythrin; o-phthaldialdehyde; pyreneand derivatives (e.g., pyrene, pyrene butyrate, and succinimidyl1-pyrene); butyrate quantum dots; Reactive Red 4 (CIBACRON™ BrilliantRed 3B-A); rhodamine and derivatives (e.g., 6-carboxy-X-rhodamine (ROX),6-carboxyrhodamine (R6G), lissamine rhodamine B sulfonyl chloriderhodarnine (Rhod), rhodamine B, rhodamine 123, rhodamine Xisothiocyanate, sulforhodamine B, sulforhodamine 101, sulfonyl chloridederivative of sulforhodamine 101 (Texas Red),N,N,N′,N′tetramethyl-6-carboxyrhodamine (TAMRA) tetramethyl rhodamine,and tetramethyl rhodamine isothiocyanate (TRITC)); riboflavin; rosolicacid; terbium chelate derivatives; Cyanine-3 (Cy3); Cyanine-5 (Cy5);cyanine-5.5 (Cy5.5), Cyanine-7 (Cy7); IRD 700; IRD 800; Alexa 647; LaJolta Blue; phthalo cyanine; and naphthalo cyanine.

In some embodiments, the detectable agent may be a non-detectablepre-cursor that becomes detectable upon activation (e.g., fluorogenictetrazine-fluorophore constructs (e.g., tetrazine-BODIPY FL,tetrazine-Oregon Green 488, or tetrazine-BODIPY TMR-X) or enzymeactivatable fluorogenic agents (e.g., PROSENSE® (VisEn Medical))). Invitro assays in which the enzyme labeled compositions can be usedinclude, but are not limited to, enzyme linked immunosorbent assays(ELISAs), immunoprecipitation assays, immunofluorescence, enzymeimmunoassays (EIA), radioimmunoassays (MA), and Western blot analysis.

Combinations

The polynucleotides, primary constructs or mmRNA may be used incombination with one or more other therapeutic, prophylactic,diagnostic, or imaging agents. By “in combination with,” it is notintended to imply that the agents must be administered at the same timeand/or formulated for delivery together, although these methods ofdelivery are within the scope of the present disclosure. Compositionscan be administered concurrently with, prior to, or subsequent to, oneor more other desired therapeutics or medical procedures. In general,each agent will be administered at a dose and/or on a time scheduledetermined for that agent. In some embodiments, the present disclosureencompasses the delivery of pharmaceutical, prophylactic, diagnostic, orimaging compositions in combination with agents that may improve theirbioavailability, reduce and/or modify their metabolism, inhibit theirexcretion, and/or modify their distribution within the body. As anon-limiting example, the polynucleotides, primary constructs and/ormmRNA may be used in combination with a pharmaceutical agent for thetreatment of cancer or to control hyperproliferative cells. In U.S. Pat.No. 7,964,571, herein incorporated by reference in its entirety, acombination therapy for the treatment of solid primary or metastasizedtumor is described using a pharmaceutical composition including a DNAplasmid encoding for interleukin-12 with a lipopolymer and alsoadministering at least one anticancer agent or chemotherapeutic.Further, the polynucleotides, primary constructs and/or mmRNA of thepresent invention that encodes anti-proliferative molecules may be in apharmaceutical composition with a lipopolymer (see e.g., U.S. Pub. No.20110218231, herein incorporated by reference in its entirety, claiminga pharmaceutical composition comprising a DNA plasmid encoding ananti-proliferative molecule and a lipopolymer) which may be administeredwith at least one chemotherapeutic or anticancer agent.

Dosing

The present invention provides methods comprising administeringpolynucleotides, primary constructs and/or mmRNA and their encodedproteins or complexes in accordance with the invention to a subject inneed thereof nucleic acids, proteins or complexes, or pharmaceutical,imaging, diagnostic, or prophylactic compositions thereof, may beadministered to a subject using any amount and any route ofadministration effective for preventing, treating, diagnosing, orimaging a disease, disorder, and/or condition (e.g., a disease,disorder, and/or condition relating to working memory deficits). Theexact amount required will vary from subject to subject, depending onthe species, age, and general condition of the subject, the severity ofthe disease, the particular composition, its mode of administration, itsmode of activity, and the like. Compositions in accordance with theinvention are typically formulated in dosage unit form for ease ofadministration and uniformity of dosage. It will be understood, however,that the total daily usage of the compositions of the present inventionmay be decided by the attending physician within the scope of soundmedical judgment. The specific therapeutically effective,prophylactically effective, or appropriate imaging dose level for anyparticular patient will depend upon a variety of factors including thedisorder being treated and the severity of the disorder; the activity ofthe specific compound employed; the specific composition employed; theage, body weight, general health, sex and diet of the patient; the timeof administration, route of administration, and rate of excretion of thespecific compound employed; the duration of the treatment; drugs used incombination or coincidental with the specific compound employed; andlike factors well known in the medical arts.

In certain embodiments, compositions in accordance with the presentinvention may be administered at dosage levels sufficient to deliverfrom about 0.0001 mg/kg to about 100 mg/kg, from about 0.001 mg/kg toabout 0.05 mg/kg, from about 0.005 mg/kg to about 0.05 mg/kg, from about0.001 mg/kg to about 0.005 mg/kg, from about 0.05 mg/kg to about 0.5mg/kg, from about 0.01 mg/kg to about 50 mg/kg, from about 0.1 mg/kg toabout 40 mg/kg, from about 0.5 mg/kg to about 30 mg/kg, from about 0.01mg/kg to about 10 mg/kg, from about 0.1 mg/kg to about 10 mg/kg, or fromabout 1 mg/kg to about 25 mg/kg, of subject body weight per day, one ormore times a day, to obtain the desired therapeutic, diagnostic,prophylactic, or imaging effect. The desired dosage may be deliveredthree times a day, two times a day, once a day, every other day, everythird day, every week, every two weeks, every three weeks, or every fourweeks. In certain embodiments, the desired dosage may be delivered usingmultiple administrations (e.g., two, three, four, five, six, seven,eight, nine, ten, eleven, twelve, thirteen, fourteen, or moreadministrations).

According to the present invention, it has been discovered thatadministration of mmRNA in split-dose regimens produce higher levels ofproteins in mammalian subjects. As used herein, a “split dose” is thedivision of single unit dose or total daily dose into two or more doses,e.g, two or more administrations of the single unit dose. As usedherein, a “single unit dose” is a dose of any therapeutic administed inone dose/at one time/single route/single point of contact, i.e., singleadministration event. As used herein, a “total daily dose” is an amountgiven or prescribed in 24 hr period. It may be administered as a singleunit dose. In one embodiment, the mmRNA of the present invention areadministed to a subject in split doses. The mmRNA may be formulated inbuffer only or in a formulation described herein.

Dosage Forms

A pharmaceutical composition described herein can be formulated into adosage form described herein, such as a topical, intranasal,intratracheal, or injectable (e.g., intravenous, intraocular,intravitreal, intramuscular, intracardiac, intraperitoneal,subcutaneous).

Liquid Dosage Forms

Liquid dosage forms for parenteral administration include, but are notlimited to, pharmaceutically acceptable emulsions, microemulsions,solutions, suspensions, syrups, and/or elixirs. In addition to activeingredients, liquid dosage forms may comprise inert diluents commonlyused in the art including, but not limited to, water or other solvents,solubilizing agents and emulsifiers such as ethyl alcohol, isopropylalcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzylbenzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils(in particular, cottonseed, groundnut, corn, germ, olive, castor, andsesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycolsand fatty acid esters of sorbitan, and mixtures thereof. In certainembodiments for parenteral administration, compositions may be mixedwith solubilizing agents such as CREMOPHOR®, alcohols, oils, modifiedoils, glycols, polysorbates, cyclodextrins, polymers, and/orcombinations thereof.

Injectable

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known art andmay include suitable dispersing agents, wetting agents, and/orsuspending agents. Sterile injectable preparations may be sterileinjectable solutions, suspensions, and/or emulsions in nontoxicparenterally acceptable diluents and/or solvents, for example, asolution in 1,3-butanediol. Among the acceptable vehicles and solventsthat may be employed include, but are not limited to, are water,Ringer's solution, U.S.P., and isotonic sodium chloride solution.Sterile, fixed oils are conventionally employed as a solvent orsuspending medium. For this purpose any bland fixed oil can be employedincluding synthetic mono- or diglycerides. Fatty acids such as oleicacid can be used in the preparation of injectables.

Injectable formulations can be sterilized, for example, by filtrationthrough a bacterial-retaining filter, and/or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium prior to use.

In order to prolong the effect of an active ingredient, it may bedesirable to slow the absorption of the active ingredient fromsubcutaneous or intramuscular injection. This may be accomplished by theuse of a liquid suspension of crystalline or amorphous material withpoor water solubility. The rate of absorption of the polynucleotide,primary construct or mmRNA then depends upon its rate of dissolutionwhich, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally administeredpolynucleotide, primary construct or mmRNA may be accomplished bydissolving or suspending the polynucleotide, primary construct or mmRNAin an oil vehicle. Injectable depot forms are made by formingmicroencapsule matrices of the polynucleotide, primary construct ormmRNA in biodegradable polymers such as polylactide-polyglycolide.Depending upon the ratio of polynucleotide, primary construct or mmRNAto polymer and the nature of the particular polymer employed, the rateof polynucleotide, primary construct or mmRNA release can be controlled.Examples of other biodegradable polymers include, but are not limitedto, poly(orthoesters) and poly(anhydrides). Depot injectableformulations may be prepared by entrapping the polynucleotide, primaryconstruct or mmRNA in liposomes or microemulsions which are compatiblewith body tissues.

Pulmonary

Formulations described herein as being useful for pulmonary delivery mayalso be use for intranasal delivery of a pharmaceutical composition.Another formulation suitable for intranasal administration may be acoarse powder comprising the active ingredient and having an averageparticle from about 0.2 μm to 500 μm. Such a formulation may beadministered in the manner in which snuff is taken, i.e. by rapidinhalation through the nasal passage from a container of the powder heldclose to the nose.

Formulations suitable for nasal administration may, for example,comprise from about as little as 0.1% (w/w) and as much as 100% (w/w) ofactive ingredient, and may comprise one or more of the additionalingredients described herein. A pharmaceutical composition may beprepared, packaged, and/or sold in a formulation suitable for buccaladministration. Such formulations may, for example, be in the form oftablets and/or lozenges made using conventional methods, and may, forexample, contain about 0.1% to 20% (w/w) active ingredient, where thebalance may comprise an orally dissolvable and/or degradable compositionand, optionally, one or more of the additional ingredients describedherein. Alternately, formulations suitable for buccal administration maycomprise a powder and/or an aerosolized and/or atomized solution and/orsuspension comprising active ingredient. Such powdered, aerosolized,and/or aerosolized formulations, when dispersed, may have an averageparticle and/or droplet size in the range from about 0.1 nm to about 200nm, and may further comprise one or more of any additional ingredientsdescribed herein.

General considerations in the formulation and/or manufacture ofpharmaceutical agents may be found, for example, in Remington: TheScience and Practice of Pharmacy 21^(st) ed., Lippincott Williams &Wilkins, 2005 (incorporated herein by reference).

Coatings or Shells

Solid dosage forms of tablets, dragees, capsules, pills, and granulescan be prepared with coatings and shells such as enteric coatings andother coatings well known in the pharmaceutical formulating art. Theymay optionally comprise opacifying agents and can be of a compositionthat they release the active ingredient(s) only, or preferentially, in acertain part of the intestinal tract, optionally, in a delayed manner.Examples of embedding compositions which can be used include polymericsubstances and waxes. Solid compositions of a similar type may beemployed as fillers in soft and hard-filled gelatin capsules using suchexcipients as lactose or milk sugar as well as high molecular weightpolyethylene glycols and the like.

Properties of Pharmaceutical Compositions

The pharmaceutical compositions described herein can be characterized byone or more of bioavailability, therapeutic window and/or volume ofdistribution.

Bioavailability

The polynucleotides, primary constructs or mmRNA, when formulated into acomposition with a delivery agent as described herein, can exhibit anincrease in bioavailability as compared to a composition lacking adelivery agent as described herein. As used herein, the term“bioavailability” refers to the systemic availability of a given amountof polynucleotides, primary constructs or mmRNA administered to amammal. Bioavailability can be assessed by measuring the area under thecurve (AUC) or the maximum serum or plasma concentration (C_(max)) ofthe unchanged form of a compound following administration of thecompound to a mammal. AUC is a determination of the area under the curveplotting the serum or plasma concentration of a compound along theordinate (Y-axis) against time along the abscissa (X-axis). Generally,the AUC for a particular compound can be calculated using methods knownto those of ordinary skill in the art and as described in G. S. Banker,Modern Pharmaceutics, Drugs and the Pharmaceutical Sciences, v. 72,Marcel Dekker, New York, Inc., 1996, herein incorporated by reference.

The C_(max) value is the maximum concentration of the compound achievedin the serum or plasma of a mammal following administration of thecompound to the mammal. The C_(max) value of a particular compound canbe measured using methods known to those of ordinary skill in the art.The phrases “increasing bioavailability” or “improving thepharmacokinetics,” as used herein mean that the systemic availability ofa first polynucleotide, primary construct or mmRNA, measured as AUC,C_(max), or C_(min) in a mammal is greater, when co-administered with adelivery agent as described herein, than when such co-administrationdoes not take place. In some embodiments, the bioavailability of thepolynucleotide, primary construct or mmRNA can increase by at leastabout 2%, at least about 5%, at least about 10%, at least about 15%, atleast about 20%, at least about 25%, at least about 30%, at least about35%, at least about 40%, at least about 45%, at least about 50%, atleast about 55%, at least about 60%, at least about 65%, at least about70%, at least about 75%, at least about 80%, at least about 85%, atleast about 90%, at least about 95%, or about 100%.

Therapeutic Window

The polynucleotides, primary constructs or mmRNA, when formulated into acomposition with a delivery agent as described herein, can exhibit anincrease in the therapeutic window of the administered polynucleotide,primary construct or mmRNA composition as compared to the therapeuticwindow of the administered polynucleotide, primary construct or mmRNAcomposition lacking a delivery agent as described herein. As used herein“therapeutic window” refers to the range of plasma concentrations, orthe range of levels of therapeutically active substance at the site ofaction, with a high probability of eliciting a therapeutic effect. Insome embodiments, the therapeutic window of the polynucleotide, primaryconstruct or mmRNA when co-administered with a delivery agent asdescribed herein can increase by at least about 2%, at least about 5%,at least about 10%, at least about 15%, at least about 20%, at leastabout 25%, at least about 30%, at least about 35%, at least about 40%,at least about 45%, at least about 50%, at least about 55%, at leastabout 60%, at least about 65%, at least about 70%, at least about 75%,at least about 80%, at least about 85%, at least about 90%, at leastabout 95%, or about 100%.

Volume of Distribution

The polynucleotides, primary constructs or mmRNA, when formulated into acomposition with a delivery agent as described herein, can exhibit animproved volume of distribution (V_(dist)), e.g., reduced or targeted,relative to a composition lacking a delivery agent as described herein.The volume of distribution (V_(dist)) relates the amount of the drug inthe body to the concentration of the drug in the blood or plasma. Asused herein, the term “volume of distribution” refers to the fluidvolume that would be required to contain the total amount of the drug inthe body at the same concentration as in the blood or plasma: V_(dist)equals the amount of drug in the body/concentration of drug in blood orplasma. For example, for a 10 mg dose and a plasma concentration of 10mg/L, the volume of distribution would be 1 liter. The volume ofdistribution reflects the extent to which the drug is present in theextravascular tissue. A large volume of distribution reflects thetendency of a compound to bind to the tissue components compared withplasma protein binding. In a clinical setting, V_(dist) can be used todetermine a loading dose to achieve a steady state concentration. Insome embodiments, the volume of distribution of the polynucleotide,primary construct or mmRNA when co-administered with a delivery agent asdescribed herein can decrease at least about 2%, at least about 5%, atleast about 10%, at least about 15%, at least about 20%, at least about25%, at least about 30%, at least about 35%, at least about 40%, atleast about 45%, at least about 50%, at least about 55%, at least about60%, at least about 65%, at least about 70%.

Biological Effect

In one embodiment, the biological effect of the modified mRNA deliveredto the animals may be categorized by analyzing the protein expression inthe animals. The reprogrammed protein expression may be determined fromanalyzing a biological sample collected from a mammal administered themodified mRNA of the present invention. In one embodiment, theexpression protein encoded by the modified mRNA administered to themammal of at least 50 pg/ml may be preferred. For example, a proteinexpression of 50-200 pg/ml for the protein encoded by the modified mRNAdelivered to the mammal may be seen as a therapeutically effectiveamount of protein in the mammal.

Detection of Modified Nucleic Acids by Mass Spectrometry

Mass spectrometry (MS) is an analytical technique that can providestructural and molecular mass/concentration information on moleculesafter their conversion to ions. The molecules are first ionized toacquire positive or negative charges and then they travel through themass analyzer to arrive at different areas of the detector according totheir mass/charge (m/z) ratio.

Mass spectrometry is performed using a mass spectrometer which includesan ion source for ionizing the fractionated sample and creating chargedmolecules for further analysis. For example ionization of the sample maybe performed by electrospray ionization (ESI), atmospheric pressurechemical ionization (APCI), photoionization, electron ionization, fastatom bombardment (FAB)/liquid secondary ionization (LSIMS), matrixassisted laser desorption/ionization (MALDI), field ionization, fielddesorption, thermospray/plasmaspray ionization, and particle beamionization. The skilled artisan will understand that the choice ofionization method can be determined based on the analyte to be measured,type of sample, the type of detector, the choice of positive versusnegative mode, etc.

After the sample has been ionized, the positively charged or negativelycharged ions thereby created may be analyzed to determine amass-to-charge ratio (i.e., m/z). Suitable analyzers for determiningmass-to-charge ratios include quadropole analyzers, ion traps analyzers,and time-of-flight analyzers. The ions may be detected using severaldetection modes. For example, selected ions may be detected (i.e., usinga selective ion monitoring mode (SIM)), or alternatively, ions may bedetected using a scanning mode, e.g., multiple reaction monitoring (MRM)or selected reaction monitoring (SRM).

Liquid chromatography-multiple reaction monitoring (LC-MS/MRM) coupledwith stable isotope labeled dilution of peptide standards has been shownto be an effective method for protein verification (e.g., Keshishian etal., Mol Cell Proteomics 2009 8: 2339-2349; Kuhn et al., Clin Chem 200955:1108-1117; Lopez et al., Clin Chem 2010 56:281-290). Unlikeuntargeted mass spectrometry frequently used in biomarker discoverystudies, targeted MS methods are peptide sequence-based modes of MS thatfocus the full analytical capacity of the instrument on tens to hundredsof selected peptides in a complex mixture. By restricting detection andfragmentation to only those peptides derived from proteins of interest,sensitivity and reproducibility are improved dramatically compared todiscovery-mode MS methods. This method of mass spectrometry-basedmultiple reaction monitoring (MRM) quantitation of proteins candramatically impact the discovery and quantitation of biomarkers viarapid, targeted, multiplexed protein expression profiling of clinicalsamples.

In one embodiment, a biological sample which may contain at least oneprotein encoded by at least one modified mRNA of the present inventionmay be analyzed by the method of MRM-MS. The quantification of thebiological sample may further include, but is not limited to,isotopically labeled peptides or proteins as internal standards.

According to the present invention, the biological sample, once obtainedfrom the subject, may be subjected to enzyme digestion. As used herein,the term “digest” means to break apart into shorter peptides. As usedherein, the phrase “treating a sample to digest proteins” meansmanipulating a sample in such a way as to break down proteins in asample. These enzymes include, but are not limited to, trypsin,endoproteinase Glu-C and chymotrypsin. In one embodiment, a biologicalsample which may contain at least one protein encoded by at least onemodified mRNA of the present invention may be digested using enzymes.

In one embodiment, a biological sample which may contain protein encodedby modified mRNA of the present invention may be analyzed for proteinusing electrospray ionization. Electrospray ionization (ESI) massspectrometry (ESIMS) uses electrical energy to aid in the transfer ofions from the solution to the gaseous phase before they are analyzed bymass spectrometry. Samples may be analyzed using methods known in theart (e.g., Ho et al., Clin Biochem Rev. 2003 24(1):3-12). The ionicspecies contained in solution may be transferred into the gas phase bydispersing a fine spray of charge droplets, evaporating the solvent andejecting the ions from the charged droplets to generate a mist of highlycharged droplets. The mist of highly charged droplets may be analyzedusing at least 1, at least 2, at least 3 or at least 4 mass analyzerssuch as, but not limited to, a quadropole mass analyzer. Further, themass spectrometry method may include a purification step. As anon-limiting example, the first quadrapole may be set to select a singlem/z ratio so it may filter out other molecular ions having a differentm/z ratio which may eliminate complicated and time-consuming samplepurification procedures prior to MS analysis.

In one embodiment, a biological sample which may contain protein encodedby modified mRNA of the present invention may be analyzed for protein ina tandem ESIMS system (e.g., MS/MS). As non-limiting examples, thedroplets may be analyzed using a product scan (or daughter scan) aprecursor scan (parent scan) a neutral loss or a multiple reactionmonitoring.

In one embodiment, a biological sample which may contain protein encodedby modified mRNA of the present invention may be analyzed usingmatrix-assisted laser desorption/ionization (MALDI) mass spectrometry(MALDIMS). MALDI provides for the nondestructive vaporization andionization of both large and small molecules, such as proteins. In MALDIanalysis, the analyte is first co-crystallized with a large molar excessof a matrix compound, which may also include, but is not limited to, anultraviolet absorbing weak organic acid. Non-limiting examples ofmatrices used in MALDI are α-cyano-4-hydroxycinnamic acid,3,5-dimethoxy-4-hydroxycinnamic acid and 2,5-dihydroxybenzoic acid.Laser radiation of the analyte-matrix mixture may result in thevaporization of the matrix and the analyte. The laser induced desorptionprovides high ion yields of the intact analyte and allows formeasurement of compounds with high accuracy. Samples may be analyzedusing methods known in the art (e.g., Lewis, Wei and Siuzdak,Encyclopedia of Analytical Chemistry 2000:5880-5894). As non-limitingexamples, mass analyzers used in the MALDI analysis may include a lineartime-of-flight (TOF), a TOF reflectron or a Fourier transform massanalyzer.

In one embodiment, the analyte-matrix mixture may be formed using thedried-droplet method. A biologic sample is mixed with a matrix to createa saturated matrix solution where the matrix-to-sample ratio isapproximately 5000:1. An aliquot (approximately 0.5-2.0 uL) of thesaturated matrix solution is then allowed to dry to form theanalyte-matrix mixture.

In one embodiment, the analyte-matrix mixture may be formed using thethin-layer method. A matrix homogeneous film is first formed and thenthe sample is then applied and may be absorbed by the matrix to form theanalyte-matrix mixture.

In one embodiment, the analyte-matrix mixture may be formed using thethick-layer method. A matrix homogeneous film is formed with anitro-cellulose matrix additive. Once the uniform nitro-cellulose matrixlayer is obtained the sample is applied and absorbed into the matrix toform the analyte-matrix mixture.

In one embodiment, the analyte-matrix mixture may be formed using thesandwich method. A thin layer of matrix crystals is prepared as in thethin-layer method followed by the addition of droplets of aqueoustrifluoroacetic acid, the sample and matrix. The sample is then absorbedinto the matrix to form the analyte-matrix mixture.

V. Uses of Polynucleotides, Primary Constructs and mmRNA of theInvention

The polynucleotides, primary constructs and mmRNA of the presentinvention may be used to alter the phenotype of cells. Thepolynucleotides, primary constructs and mmRNA of the invention mayencode peptides, polypeptides or multiple proteins to producepolypeptides of interest. The polypeptides of interest may be used intherapeutics and/or clinical and research settings. As a non-limitingexample, the polypeptides of interest may include reprogramming factors,differentiation factors and de-differentiation factors.

Therapeutics Therapeutic Agents

The polynucleotides, primary constructs or mmRNA of the presentinvention, such as modified nucleic acids and modified RNAs, and theproteins translated from them described herein can be used astherapeutic or prophylactic agents. They are provided for use inmedicine, therapy and preventative treatments. For example, apolynucleotide, primary construct or mmRNA described herein can beadministered to a subject, wherein the polynucleotide, primary constructor mmRNA is translated in vivo to produce a therapeutic or prophylacticpolypeptide in the subject. Provided are compositions, methods, kits,and reagents for diagnosis, treatment or prevention of a disease orcondition in humans and other mammals. The active therapeutic agents ofthe invention include polynucleotides, primary constructs or mmRNA,cells containing the polynucleotides, primary constructs or mmRNA orpolypeptides translated from the polynucleotides, primary constructs ormmRNA.

In certain embodiments, provided herein are combination therapeuticscontaining one or more polynucleotide, primary construct or mmRNAcontaining translatable regions that encode for a protein or proteins.

Provided herein are methods of inducing translation of a recombinantpolypeptide in a cell population using the polynucleotide, primaryconstruct or mmRNA described herein. Such translation can be in vivo, exvivo, in culture, or in vitro. The cell population is contacted with aneffective amount of a composition containing a nucleic acid that has atleast one nucleoside modification, and a translatable region encodingthe recombinant polypeptide. The population is contacted underconditions such that the nucleic acid is localized into one or morecells of the cell population and the recombinant polypeptide istranslated in the cell from the nucleic acid.

An “effective amount” of the composition is provided based, at least inpart, on the target tissue, target cell type, means of administration,physical characteristics of the nucleic acid (e.g., size, and extent ofmodified nucleosides), and other determinants. In general, an effectiveamount of the composition provides efficient protein production in thecell, preferably more efficient than a composition containing acorresponding unmodified nucleic acid. Increased efficiency may bedemonstrated by increased cell transfection (i.e., the percentage ofcells transfected with the nucleic acid), increased protein translationfrom the nucleic acid, decreased nucleic acid degradation (asdemonstrated, e.g., by increased duration of protein translation from amodified nucleic acid), or reduced innate immune response of the hostcell.

Aspects of the invention are directed to methods of inducing in vivotranslation of a recombinant polypeptide in a mammalian subject in needthereof. Therein, an effective amount of a composition containing anucleic acid that has at least one structural or chemical modificationand a translatable region encoding the recombinant polypeptide isadministered to the subject using the delivery methods described herein.The nucleic acid is provided in an amount and under other conditionssuch that the nucleic acid is localized into a cell of the subject andthe recombinant polypeptide is translated in the cell from the nucleicacid. The cell in which the nucleic acid is localized, or the tissue inwhich the cell is present, may be targeted with one or more than onerounds of nucleic acid administration.

In certain embodiments, the administered polynucleotide, primaryconstruct or mmRNA directs production of one or more recombinantpolypeptides that provide a functional activity which is substantiallyabsent in the cell, tissue or organism in which the recombinantpolypeptide is translated. For example, the missing functional activitymay be enzymatic, structural, or gene regulatory in nature. In relatedembodiments, the administered polynucleotide, primary construct or mmRNAdirects production of one or more recombinant polypeptides thatincreases (e.g., synergistically) a functional activity which is presentbut substantially deficient in the cell in which the recombinantpolypeptide is translated.

In other embodiments, the administered polynucleotide, primary constructor mmRNA directs production of one or more recombinant polypeptides thatreplace a polypeptide (or multiple polypeptides) that is substantiallyabsent in the cell in which the recombinant polypeptide is translated.Such absence may be due to genetic mutation of the encoding gene orregulatory pathway thereof. In some embodiments, the recombinantpolypeptide increases the level of an endogenous protein in the cell toa desirable level; such an increase may bring the level of theendogenous protein from a subnormal level to a normal level or from anormal level to a super-normal level.

Alternatively, the recombinant polypeptide functions to antagonize theactivity of an endogenous protein present in, on the surface of, orsecreted from the cell. Usually, the activity of the endogenous proteinis deleterious to the subject; for example, due to mutation of theendogenous protein resulting in altered activity or localization.Additionally, the recombinant polypeptide antagonizes, directly orindirectly, the activity of a biological moiety present in, on thesurface of, or secreted from the cell. Examples of antagonizedbiological moieties include lipids (e.g., cholesterol), a lipoprotein(e.g., low density lipoprotein), a nucleic acid, a carbohydrate, aprotein toxin such as shiga and tetanus toxins, or a small moleculetoxin such as botulinum, cholera, and diphtheria toxins. Additionally,the antagonized biological molecule may be an endogenous protein thatexhibits an undesirable activity, such as a cytotoxic or cytostaticactivity.

The recombinant proteins described herein may be engineered forlocalization within the cell, potentially within a specific compartmentsuch as the nucleus, or are engineered for secretion from the cell ortranslocation to the plasma membrane of the cell.

Other aspects of the present disclosure relate to transplantation ofcells containing polynucleotide, primary construct, or mmRNA to amammalian subject. Administration of cells to mammalian subjects isknown to those of ordinary skill in the art, and include, but is notlimited to, local implantation (e.g., topical or subcutaneousadministration), organ delivery or systemic injection (e.g., intravenousinjection or inhalation), and the formulation of cells inpharmaceutically acceptable carrier. Such compositions containingpolynucleotide, primary construct, or mmRNA can be formulated foradministration intramuscularly, transarterially, intraperitoneally,intravenously, intranasally, subcutaneously, endoscopically,transdermally, or intrathecally. In some embodiments, the compositionmay be formulated for extended release. The subject to whom thetherapeutic agent may be administered suffers from or may be at risk ofdeveloping a disease, disorder, or deleterious condition. Provided aremethods of identifying, diagnosing, and classifying subjects on thesebases, which may include clinical diagnosis, biomarker levels,genome-wide association studies (GWAS), and other methods known in theart.

Diseases or Disorders Familial Hypercholesterolemia

In one embodiment, the polynucleotide, primary construct, or mmRNA ofthe present invention may be used to treat familial hypercholesterolemia(FH). As used herein, the term “familial hypercholesterolemia” or “FH”refers to an autosomal dominant genetic disorder characterized byelevated levels of low density lipoprotein (LDL)-associated cholesterolin the plasma. Compared with LDL cholesterol levels in normal patients(e.g., <130 mg/dL), levels in heterozygous and homozygous FH patientsoften rise to 350-550 mg/dL and to >600 mg/dL, respectively. Elevationin LDL cholesterol at these levels in patients or subjects with FH leadsto cholesterol deposition within tissues and may have an increased riskfor cardiovascular disease at a young age. In some embodiments, highlevels of LDL in the blood of these individuals may be the result ofmutations in the gene encoding the LDL receptor. LDL is produced withinthe circulation by lipolytic catabolism of triglyeride-rich very lowdensity lipoproteins or VLDL. Following lipid transfer andesterification reactions, it is believed, and is no means limiting, thatthe LDL receptor binds LDL in the circulation and facilitatesendocytosis of LDL into the hepatic cell surface that the receptor isexpressed on. When this receptor is dysfunctional, LDL levels remainelevated in the circulation during to their prolonged retention in thebloodstream and promote the development of atherosclerosis. Inactivatingmutations in the LDLR gene are responsible for the majority of FH caseswith LDLR expression in heterogygous and homozygous patients generally˜50% and ˜10-15% of normal, respectively. Individuals with FH may beheterozygous or homozygous for FH-related gene mutations. HeterozygousFH is one of the most common genetic disorders with a prevelance of˜1/500 in the general population; while homozygous forms of the diseaseare more rare with a prevelance of ˜1/1,000,000. Symptoms in homozygousindividuals can be more severe. Diagnosis is possible during childhoodor young adulthood by methods known in the art including, but notlimited to, a physical exam that reveals xanthomas (fatty skin growths).Earlier diagnosis of FH may be made through an analysis of familyhistory and genetics. (Sjouke, B. et al., Familial hypercholesterolemia:present and future management. Curr Cardiol Rep. 2011 December;13(6):527-36; Avis, H. J. et al., A systematic review and meta-analysisof statin therapy in children with familial hypercholesterolemia.Arterioscler Thromb Vasc Biol. 2007 August; 27(8):1803-10; each of whichare herein incorporated by reference in their entireties).

Current therapeutic agents, such as statins, Niacin and resins, canreduce serum cholesterol levels, either directly or indirectly, throughinduction of LDLR expression in the liver. While effective for manyheterozygous FH patients, these approaches can be problematic. At least25-30% of patients taking these drugs fail to achieve their desired LDLcholesterol goals. These agents can be even less effective in treatinghomozygous FH primarily due to the low residual levels of functionalLDLRs in the liver of these patients. Most of these patients arenon-responsive to statins and in severe forms of disease, treatment islimited to LDL apheresis and liver transplantation. Current treatmentsare not always successful in lowering LDL-Cholesterol levels to target;therefore, new treatments are urgently needed.

In one embodiment, patients with FH may be administered a compositioncomprising at least one polynucleotide, primary construct or mmRNA ofthe present invention. The polynucleotide, primary construct or mmRNAmay encode a peptide, protein or fragment thereof such as, but notlimited to, low density lipoprotein receptor (LDLR), apolipoprotein B(APOB), and proprotein convertase subtilisin/kexin type 9 (PCSK9).

In one embodiment, FH may be treated by administering a composition ofthe present invention comprising at least one polynucleotide, primaryconstruct or mmRNA encoding a peptide, protein or fragment thereof ofLDLR. In another embodiment, FH may be treated by administering acomposition of the present invention comprising at least onepolynucleotide, primary construct or mmRNA encoding a peptide, proteinor fragment thereof.

In one embodiment, FH may be treated by administering a composition ofthe present invention comprising at least one polynucleotide, primaryconstruct or mmRNA encoding a peptide, protein or fragment thereof ofAPOB. In another embodiment, FH may be treated by administering acomposition of the present invention comprising at least onepolynucleotide, primary construct or mmRNA encoding a peptide, proteinor fragment thereof. In one embodiment, FH may be treated byadministering a composition of the present invention comprising at leastone polynucleotide, primary construct or mmRNA encoding a peptide,protein or fragment thereof of PCSK9. In another embodiment, FH may betreated by administering a composition of the present inventioncomprising at least one polynucleotide, primary construct or mmRNAencoding a peptide, protein or fragment thereof.

In another embodiment, it may be useful to optimize the expression of aspecific polypeptide in a cell line or collection of cell lines ofpotential interest, particularly a polypeptide of interest such as aprotein variant of a reference protein having a known activity. In oneembodiment, provided is a method of optimizing expression of apolypeptide of interest in a target cell, by providing a plurality oftarget cell types, and independently contacting with each of theplurality of target cell types a modified mRNA encoding a polypeptide.Additionally, culture conditions may be altered to increase proteinproduction efficiency. Subsequently, the presence and/or level of thepolypeptide of interest in the plurality of target cell types isdetected and/or quantitated, allowing for the optimization of apolypeptide of interest's expression by selection of an efficient targetcell and cell culture conditions relating thereto. Such methods may beuseful when the polypeptide of interest contains one or morepost-translational modifications or has substantial tertiary structure,which often complicate efficient protein production.

Methods and compositions described herein may be used to produceproteins which are capable of attenuating or blocking the endogenousagonist biological response and/or antagonizing a receptor or signalingmolecule in a mammalian subject. For example, IL-12 and IL-23 receptorsignaling may be enhanced in chronic autoimmune disorders such asmultiple sclerosis and inflammatory diseases such as rheumatoidarthritis, psoriasis, lupus erythematosus, ankylosing spondylitis andChron's disease (Kikly K, Liu L, Na S, Sedgwich J D (2006) Cur. Opin.Immunol. 18(6): 670-5). In another embodiment, a nucleic acid encodes anantagonist for chemokine receptors. Chemokine receptors CXCR-4 and CCR-5are required for HIV enry into host cells (Arenzana-Seisdedos F et al,(1996) Nature. October 3; 383 (6599):400).

Expression of Ligand or Receptor on Cell Surface

In some aspects and embodiments of the aspects described herein, thepolynucleotides, primary constructs or mmRNA described herein can beused to express a ligand or ligand receptor on the surface of a cell(e.g., a homing moiety). A ligand or ligand receptor moiety attached toa cell surface can permit the cell to have a desired biologicalinteraction with a tissue or an agent in vivo. A ligand can be anantibody, an antibody fragment, an aptamer, a peptide, a vitamin, acarbohydrate, a protein or polypeptide, a receptor, e.g., cell-surfacereceptor, an adhesion molecule, a glycoprotein, a sugar residue, atherapeutic agent, a drug, a glycosaminoglycan, or any combinationthereof. For example, a ligand can be an antibody that recognizes acancer-cell specific antigen, rendering the cell capable ofpreferentially interacting with tumor cells to permit tumor-specificlocalization of a modified cell. A ligand can confer the ability of acell composition to accumulate in a tissue to be treated, since apreferred ligand may be capable of interacting with a target molecule onthe external face of a tissue to be treated. Ligands having limitedcross-reactivity to other tissues are generally preferred.

In some cases, a ligand can act as a homing moiety which permits thecell to target to a specific tissue or interact with a specific ligand.Such homing moieties can include, but are not limited to, any member ofa specific binding pair, antibodies, monoclonal antibodies, orderivatives or analogs thereof, including without limitation: Fvfragments, single chain Fv (scFv) fragments, Fab′ fragments, F(ab′)2fragments, single domain antibodies, camelized antibodies and antibodyfragments, humanized antibodies and antibody fragments, and multivalentversions of the foregoing; multivalent binding reagents includingwithout limitation: monospecific or bispecific antibodies, such asdisulfide stabilized Fv fragments, scFv tandems ((SCFV)2 fragments),diabodies, tribodies or tetrabodies, which typically are covalentlylinked or otherwise stabilized (i.e., leucine zipper or helixstabilized) scFv fragments; and other homing moieties include forexample, aptamers, receptors, and fusion proteins.

In some embodiments, the homing moiety may be a surface-bound antibody,which can permit tuning of cell targeting specificity. This isespecially useful since highly specific antibodies can be raised againstan epitope of interest for the desired targeting site. In oneembodiment, multiple antibodies are expressed on the surface of a cell,and each antibody can have a different specificity for a desired target.Such approaches can increase the avidity and specificity of hominginteractions.

A skilled artisan can select any homing moiety based on the desiredlocalization or function of the cell, for example an estrogen receptorligand, such as tamoxifen, can target cells to estrogen-dependent breastcancer cells that have an increased number of estrogen receptors on thecell surface. Other non-limiting examples of ligand/receptorinteractions include CCRI (e.g., for treatment of inflamed joint tissuesor brain in rheumatoid arthritis, and/or multiple sclerosis), CCR7, CCR8(e.g., targeting to lymph node tissue), CCR6, CCR9,CCR10 (e.g., totarget to intestinal tissue), CCR4, CCR10 (e.g., for targeting to skin),CXCR4 (e.g., for general enhanced transmigration), HCELL (e.g., fortreatment of inflammation and inflammatory disorders, bone marrow),Alpha4beta7 (e.g., for intestinal mucosa targeting), VLA-4/VCAM-1 (e.g.,targeting to endothelium). In general, any receptor involved intargeting (e.g., cancer metastasis) can be harnessed for use in themethods and compositions described herein.

VI. Kits and Devices

The invention provides a variety of kits for conveniently and/oreffectively carrying out methods of the present invention. Typicallykits will comprise sufficient amounts and/or numbers of components toallow a user to perform multiple treatments of a subject(s) and/or toperform multiple experiments, and contact cells and/or a population ofcells at least once.

In one aspect, the present invention provides kits comprising themolecules (polynucleotides, primary constructs or mmRNA) of theinvention. In one embodiment, the kit comprises one or more functionalantibodies or function fragments thereof.

Kits and devices useful in combination with the polynucleotides, primaryconstructs or mmRNA) of the invention include those disclosed inco-pending U.S. Provisional Patent Application No. 61/737,130 filed Dec.14, 2012, the contents of which are incorporated herein by reference intheir entirety.

VII. Definitions

At various places in the present specification, substituents ofcompounds of the present disclosure are disclosed in groups or inranges. It is specifically intended that the present disclosure includeeach and every individual subcombination of the members of such groupsand ranges. For example, the term “C₁₋₆ alkyl” is specifically intendedto individually disclose methyl, ethyl, C₃ alkyl, C₄ alkyl, C₅ alkyl,and C₆ alkyl.

About: As used herein, the term “about” means+/−10% of the recitedvalue.

Administered in combination: As used herein, the term “administered incombination” or “combined administration” means that two or more agentsare administered to a subject at the same time or within an intervalsuch that there may be an overlap of an effect of each agent on thepatient. In some embodiments, they are administered within about 60, 30,15, 10, 5, or 1 minute of one another. In some embodiments, theadministrations of the agents are spaced sufficiently closely togethersuch that a combinatorial (e.g., a synergistic) effect is achieved.

Animal: As used herein, the term “animal” refers to any member of theanimal kingdom. In some embodiments, “animal” refers to humans at anystage of development. In some embodiments, “animal” refers to non-humananimals at any stage of development. In certain embodiments, thenon-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit,a monkey, a dog, a cat, a sheep, cattle, a primate, or a pig). In someembodiments, animals include, but are not limited to, mammals, birds,reptiles, amphibians, fish, and worms. In some embodiments, the animalis a transgenic animal, genetically-engineered animal, or a clone.

Approximately: As used herein, the term “approximately” or “about,” asapplied to one or more values of interest, refers to a value that issimilar to a stated reference value. In certain embodiments, the term“approximately” or “about” refers to a range of values that fall within25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%,6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than orless than) of the stated reference value unless otherwise stated orotherwise evident from the context (except where such number wouldexceed 100% of a possible value).

Associated with: As used herein, the terms “associated with,”“conjugated,” “linked,” “attached,” and “tethered,” when used withrespect to two or more moieties, means that the moieties are physicallyassociated or connected with one another, either directly or via one ormore additional moieties that serves as a linking agent, to form astructure that is sufficiently stable so that the moieties remainphysically associated under the conditions in which the structure isused, e.g., physiological conditions. An “association” need not bestrictly through direct covalent chemical bonding. It may also suggestionic or hydrogen bonding or a hybridization based connectivitysufficiently stable such that the “associated” entities remainphysically associated.

Bifunctional: As used herein, the term “bifunctional” refers to anysubstance, molecule or moiety which is capable of or maintains at leasttwo functions. The functions may effect the same outcome or a differentoutcome. The structure that produces the function may be the same ordifferent. For example, bifunctional modified RNAs of the presentinvention may encode a cytotoxic peptide (a first function) while thosenucleosides which comprise the encoding RNA are, in and of themselves,cytotoxic (second function). In this example, delivery of thebifunctional modified RNA to a cancer cell would produce not only apeptide or protein molecule which may ameliorate or treat the cancer butwould also deliver a cytotoxic payload of nucleosides to the cell shoulddegradation, instead of translation of the modified RNA, occur.

Biocompatible: As used herein, the term “biocompatible” means compatiblewith living cells, tissues, organs or systems posing little to no riskof injury, toxicity or rejection by the immune system.

Biodegradable: As used herein, the term “biodegradable” means capable ofbeing broken down into innocuous products by the action of livingthings.

Biologically active: As used herein, the phrase “biologically active”refers to a characteristic of any substance that has activity in abiological system and/or organism. For instance, a substance that, whenadministered to an organism, has a biological effect on that organism,is considered to be biologically active. In particular embodiments, apolynucleotide, primary construct or mmRNA of the present invention maybe considered biologically active if even a portion of thepolynucleotide, primary construct or mmRNA is biologically active ormimics an activity considered biologically relevant.

Cancer stem cells: As used herein, “cancer stem cells” are cells thatcan undergo self-renewal and/or abnormal proliferation anddifferentiation to form a tumor.

Chemical terms: Chemical terms not otherwise defined herein, willconform to the chemical term definitions provided in co-pending U.S.Provisional Patent Application No. 61/737,130 filed Dec. 14, 2012, thecontents of which are incorporated herein by reference in theirentirety.

The term “diastereomer,” as used herein means stereoisomers that are notmirror images of one another and are non-superimposable on one another.

The term “effective amount” of an agent, as used herein, is that amountsufficient to effect beneficial or desired results, for example,clinical results, and, as such, an “effective amount” depends upon thecontext in which it is being applied. For example, in the context ofadministering an agent that treats cancer, an effective amount of anagent is, for example, an amount sufficient to achieve treatment, asdefined herein, of cancer, as compared to the response obtained withoutadministration of the agent.

The term “enantiomer,” as used herein, means each individual opticallyactive form of a compound of the invention, having an optical purity orenantiomeric excess (as determined by methods standard in the art) of atleast 80% (i.e., at least 90% of one enantiomer and at most 10% of theother enantiomer), preferably at least 90% and more preferably at least98%.

The term “isomer,” as used herein, means any tautomer, stereoisomer,enantiomer, or diastereomer of any compound of the invention. It isrecognized that the compounds of the invention can have one or morechiral centers and/or double bonds and, therefore, exist asstereoisomers, such as double-bond isomers (i.e., geometric E/Z isomers)or diastereomers (e.g., enantiomers (i.e., (+) or (−)) or cis/transisomers). According to the invention, the chemical structures depictedherein, and therefore the compounds of the invention, encompass all ofthe corresponding stereoisomers, that is, both the stereomerically pureform (e.g., geometrically pure, enantiomerically pure, ordiastereomerically pure) and enantiomeric and stereoisomeric mixtures,e.g., racemates. Enantiomeric and stereoisomeric mixtures of compoundsof the invention can typically be resolved into their componentenantiomers or stereoisomers by well-known methods, such as chiral-phasegas chromatography, chiral-phase high performance liquid chromatography,crystallizing the compound as a chiral salt complex, or crystallizingthe compound in a chiral solvent. Enantiomers and stereoisomers can alsobe obtained from stereomerically or enantiomerically pure intermediates,reagents, and catalysts by well-known asymmetric synthetic methods.

The term “stereoisomer,” as used herein, refers to all possibledifferent isomeric as well as conformational forms which a compound maypossess (e.g., a compound of any formula described herein), inparticular all possible stereochemically and conformationally isomericforms, all diastereomers, enantiomers and/or conformers of the basicmolecular structure. Some compounds of the present invention may existin different tautomeric forms, all of the latter being included withinthe scope of the present invention.

Compound: As used herein, the term “compound,” is meant to include allstereoisomers, geometric isomers, tautomers, and isotopes of thestructures depicted.

The compounds described herein can be asymmetric (e.g., having one ormore stereocenters). All stereoisomers, such as enantiomers anddiastereomers, are intended unless otherwise indicated. Compounds of thepresent disclosure that contain asymmetrically substituted carbon atomscan be isolated in optically active or racemic forms. Methods on how toprepare optically active forms from optically active starting materialsare known in the art, such as by resolution of racemic mixtures or bystereoselective synthesis. Many geometric isomers of olefins, C═N doublebonds, and the like can also be present in the compounds describedherein, and all such stable isomers are contemplated in the presentdisclosure. Cis and trans geometric isomers of the compounds of thepresent disclosure are described and may be isolated as a mixture ofisomers or as separated isomeric forms.

Compounds of the present disclosure also include tautomeric forms.Tautomeric forms result from the swapping of a single bond with anadjacent double bond and the concomitant migration of a proton.Tautomeric forms include prototropic tautomers which are isomericprotonation states having the same empirical formula and total charge.Examples prototropic tautomers include ketone—enol pairs, amide—imidicacid pairs, lactam—lactim pairs, amide—imidic acid pairs, enamine—iminepairs, and annular forms where a proton can occupy two or more positionsof a heterocyclic system, such as, 1H- and 3H-imidazole, 1H-, 2H- and4H-1,2,4-triazole, 1H- and 2H-isoindole, and 1H- and 2H-pyrazole.Tautomeric forms can be in equilibrium or sterically locked into oneform by appropriate substitution.

Compounds of the present disclosure also include all of the isotopes ofthe atoms occurring in the intermediate or final compounds. “Isotopes”refers to atoms having the same atomic number but different mass numbersresulting from a different number of neutrons in the nuclei. Forexample, isotopes of hydrogen include tritium and deuterium.

The compounds and salts of the present disclosure can be prepared incombination with solvent or water molecules to form solvates andhydrates by routine methods.

Committed: As used herein, the term “committed” means, when referring toa cell, when the cell is far enough into the differentiation pathwaywhere, under normal circumstances, it will continue to differentiateinto a specific cell type or subset of cell type instead of into adifferent cell type or reverting to a lesser differentiated cell type.

Conserved: As used herein, the term “conserved” refers to nucleotides oramino acid residues of a polynucleotide sequence or polypeptidesequence, respectively, that are those that occur unaltered in the sameposition of two or more sequences being compared. Nucleotides or aminoacids that are relatively conserved are those that are conserved amongstmore related sequences than nucleotides or amino acids appearingelsewhere in the sequences.

In some embodiments, two or more sequences are said to be “completelyconserved” if they are 100% identical to one another. In someembodiments, two or more sequences are said to be “highly conserved” ifthey are at least 70% identical, at least 80% identical, at least 90%identical, or at least 95% identical to one another. In someembodiments, two or more sequences are said to be “highly conserved” ifthey are about 70% identical, about 80% identical, about 90% identical,about 95%, about 98%, or about 99% identical to one another. In someembodiments, two or more sequences are said to be “conserved” if theyare at least 30% identical, at least 40% identical, at least 50%identical, at least 60% identical, at least 70% identical, at least 80%identical, at least 90% identical, or at least 95% identical to oneanother. In some embodiments, two or more sequences are said to be“conserved” if they are about 30% identical, about 40% identical, about50% identical, about 60% identical, about 70% identical, about 80%identical, about 90% identical, about 95% identical, about 98%identical, or about 99% identical to one another. Conservation ofsequence may apply to the entire length of an oligonucleotide orpolypeptide or may apply to a portion, region or feature thereof.

Controlled Release: As used herein, the term “controlled release” refersto a pharmaceutical composition or compound release profile thatconforms to a particular pattern of release to effect a therapeuticoutcome.

Cyclic or Cyclized: As used herein, the term “cyclic” refers to thepresence of a continuous loop. Cyclic molecules need not be circular,only joined to form an unbroken chain of subunits. Cyclic molecules suchas the engineered RNA or mRNA of the present invention may be singleunits or multimers or comprise one or more components of a complex orhigher order structure.

Cytostatic: As used herein, “cytostatic” refers to inhibiting, reducing,suppressing the growth, division, or multiplication of a cell (e.g., amammalian cell (e.g., a human cell)), bacterium, virus, fungus,protozoan, parasite, prion, or a combination thereof.

Cytotoxic: As used herein, “cytotoxic” refers to killing or causinginjurious, toxic, or deadly effect on a cell (e.g., a mammalian cell(e.g., a human cell)), bacterium, virus, fungus, protozoan, parasite,prion, or a combination thereof.

Delivery: As used herein, “delivery” refers to the act or manner ofdelivering a compound, substance, entity, moiety, cargo or payload.

Delivery Agent: As used herein, “delivery agent” refers to any substancewhich facilitates, at least in part, the in vivo delivery of apolynucleotide, primary construct or mmRNA to targeted cells.

Destabilized: As used herein, the term “destable,” “destabilize,” or“destabilizing region” means a region or molecule that is less stablethan a starting, wild-type or native form of the same region ormolecule.

Detectable label: As used herein, “detectable label” refers to one ormore markers, signals, or moieties which are attached, incorporated orassociated with another entity that is readily detected by methods knownin the art including radiography, fluorescence, chemiluminescence,enzymatic activity, absorbance and the like. Detectable labels includeradioisotopes, fluorophores, chromophores, enzymes, dyes, metal ions,ligands such as biotin, avidin, streptavidin and haptens, quantum dots,and the like. Detectable labels may be located at any position in thepeptides or proteins disclosed herein. They may be within the aminoacids, the peptides, or proteins, or located at the N- or C-termini.

Developmental Potential: As used herein, “developmental potential” or“developmental potency” refers to the total of all developmental cellfates or cell types that can be achieved by a cell upon differentiation.

Developmental Potential Altering Factor: As used herein, “developmentalpotential altering factor” refers to a protein or RNA which can alterthe developmental potential of a cell.

Digest: As used herein, the term “digest” means to break apart intosmaller pieces or components. When referring to polypeptides orproteins, digestion results in the production of peptides.

Differentiated cell: As used herein, the term “differentiated cell”refers to any somatic cell that is not, in its native form, pluripotent.Differentiated cell also encompasses cells that are partiallydifferentiated.

Differentiation: As used herein, the term “differentiation factor”refers to a developmental potential altering factor such as a protein,RNA or small molecule that can induce a cell to differentiate to adesired cell-type.

Differentiate: As used herein, “differentiate” refers to the processwhere an uncommitted or less committed cell acquires the features of acommitted cell.

Distal: As used herein, the term “distal” means situated away from thecenter or away from a point or region of interest.

Dose splitting factor (DSF)-ratio of PUD of dose split treatment dividedby PUD of total daily dose or single unit dose. The value is derivedfrom comparison of dosing regimens groups.

Embryonic stem cell: As used herein, the term “embryonic stem cell”refers to naturally occurring pluripotent stem cells of the inner cellmass of the embryonic blastocyst.

Encapsulate: As used herein, the term “encapsulate” means to enclose,surround or encase.

Encoded protein cleavage signal: As used herein, “encoded proteincleavage signal” refers to the nucleotide sequence which encodes aprotein cleavage signal.

Engineered: As used herein, embodiments of the invention are“engineered” when they are designed to have a feature or property,whether structural or chemical, that varies from a starting point, wildtype or native molecule.

Exosome: As used herein, “exosome” is a vesicle secreted by mammaliancells or a complex involved in RNA degradation.

Expression: As used herein, “expression” of a nucleic acid sequencerefers to one or more of the following events: (1) production of an RNAtemplate from a DNA sequence (e.g., by transcription); (2) processing ofan RNA transcript (e.g., by splicing, editing, 5′ cap formation, and/or3′ end processing); (3) translation of an RNA into a polypeptide orprotein; and (4) post-translational modification of a polypeptide orprotein.

Feature: As used herein, a “feature” refers to a characteristic, aproperty, or a distinctive element.

Formulation: As used herein, a “formulation” includes at least apolynucleotide, primary construct or mmRNA and a delivery agent.

Fragment: A “fragment,” as used herein, refers to a portion. Forexample, fragments of proteins may comprise polypeptides obtained bydigesting full-length protein isolated from cultured cells.

Functional: As used herein, a “functional” biological molecule is abiological molecule in a form in which it exhibits a property and/oractivity by which it is characterized.

Homology: As used herein, the term “homology” refers to the overallrelatedness between polymeric molecules, e.g. between nucleic acidmolecules (e.g. DNA molecules and/or RNA molecules) and/or betweenpolypeptide molecules. In some embodiments, polymeric molecules areconsidered to be “homologous” to one another if their sequences are atleast 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, or 99% identical or similar. The term “homologous” necessarilyrefers to a comparison between at least two sequences (polynucleotide orpolypeptide sequences). In accordance with the invention, twopolynucleotide sequences are considered to be homologous if thepolypeptides they encode are at least about 50%, 60%, 70%, 80%, 90%,95%, or even 99% for at least one stretch of at least about 20 aminoacids. In some embodiments, homologous polynucleotide sequences arecharacterized by the ability to encode a stretch of at least 4-5uniquely specified amino acids. For polynucleotide sequences less than60 nucleotides in length, homology is determined by the ability toencode a stretch of at least 4-5 uniquely specified amino acids. Inaccordance with the invention, two protein sequences are considered tobe homologous if the proteins are at least about 50%, 60%, 70%, 80%, or90% identical for at least one stretch of at least about 20 amino acids.

Identity: As used herein, the term “identity” refers to the overallrelatedness between polymeric molecules, e.g., between oligonucleotidemolecules (e.g. DNA molecules and/or RNA molecules) and/or betweenpolypeptide molecules. Calculation of the percent identity of twopolynucleotide sequences, for example, can be performed by aligning thetwo sequences for optimal comparison purposes (e.g., gaps can beintroduced in one or both of a first and a second nucleic acid sequencesfor optimal alignment and non-identical sequences can be disregarded forcomparison purposes). In certain embodiments, the length of a sequencealigned for comparison purposes is at least 30%, at least 40%, at least50%, at least 60%, at least 70%, at least 80%, at least 90%, at least95%, or 100% of the length of the reference sequence. The nucleotides atcorresponding nucleotide positions are then compared. When a position inthe first sequence is occupied by the same nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position. The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences, taking into account the number of gaps, and the length ofeach gap, which needs to be introduced for optimal alignment of the twosequences. The comparison of sequences and determination of percentidentity between two sequences can be accomplished using a mathematicalalgorithm. For example, the percent identity between two nucleotidesequences can be determined using methods such as those described inComputational Molecular Biology, Lesk, A. M., ed., Oxford UniversityPress, New York, 1988; Biocomputing: Informatics and Genome Projects,Smith, D. W., ed., Academic Press, New York, 1993; Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press, 1987; ComputerAnalysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G.,eds., Humana Press, New Jersey, 1994; and Sequence Analysis Primer,Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991;each of which is incorporated herein by reference. For example, thepercent identity between two nucleotide sequences can be determinedusing the algorithm of Meyers and Miller (CABIOS, 1989, 4:11-17), whichhas been incorporated into the ALIGN program (version 2.0) using aPAM120 weight residue table, a gap length penalty of 12 and a gappenalty of 4. The percent identity between two nucleotide sequences can,alternatively, be determined using the GAP program in the GCG softwarepackage using an NWSgapdna.CMP matrix. Methods commonly employed todetermine percent identity between sequences include, but are notlimited to those disclosed in Carillo, H., and Lipman, D., SIAM JApplied Math., 48:1073 (1988); incorporated herein by reference.Techniques for determining identity are codified in publicly availablecomputer programs. Exemplary computer software to determine homologybetween two sequences include, but are not limited to, GCG programpackage, Devereux, J., et al., Nucleic Acids Research, 12(1), 387(1984)), BLASTP, BLASTN, and FASTA Altschul, S. F. et al., J. Molec.Biol., 215, 403 (1990)).

Inhibit expression of a gene: As used herein, the phrase “inhibitexpression of a gene” means to cause a reduction in the amount of anexpression product of the gene. The expression product can be an RNAtranscribed from the gene (e.g., an mRNA) or a polypeptide translatedfrom an mRNA transcribed from the gene. Typically a reduction in thelevel of an mRNA results in a reduction in the level of a polypeptidetranslated therefrom. The level of expression may be determined usingstandard techniques for measuring mRNA or protein.

In vitro: As used herein, the term “in vitro” refers to events thatoccur in an artificial environment, e.g., in a test tube or reactionvessel, in cell culture, in a Petri dish, etc., rather than within anorganism (e.g., animal, plant, or microbe).

In vivo: As used herein, the term “in vivo” refers to events that occurwithin an organism (e.g., animal, plant, or microbe or cell or tissuethereof).

Isolated: As used herein, the term “isolated” refers to a substance orentity that has been separated from at least some of the components withwhich it was associated (whether in nature or in an experimentalsetting). Isolated substances may have varying levels of purity inreference to the substances from which they have been associated.Isolated substances and/or entities may be separated from at least about10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%,about 80%, about 90%, or more of the other components with which theywere initially associated. In some embodiments, isolated agents are morethan about 80%, about 85%, about 90%, about 91%, about 92%, about 93%,about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, ormore than about 99% pure. As used herein, a substance is “pure” if it issubstantially free of other components.

Substantially isolated: By “substantially isolated” is meant that thecompound is substantially separated from the environment in which it wasformed or detected. Partial separation can include, for example, acomposition enriched in the compound of the present disclosure.Substantial separation can include compositions containing at leastabout 50%, at least about 60%, at least about 70%, at least about 80%,at least about 90%, at least about 95%, at least about 97%, or at leastabout 99% by weight of the compound of the present disclosure, or saltthereof. Methods for isolating compounds and their salts are routine inthe art.

Linker: As used herein, a linker refers to a group of atoms, e.g.,10-1,000 atoms, and can be comprised of the atoms or groups such as, butnot limited to, carbon, amino, alkylamino, oxygen, sulfur, sulfoxide,sulfonyl, carbonyl, and imine. The linker can be attached to a modifiednucleoside or nucleotide on the nucleobase or sugar moiety at a firstend, and to a payload, e.g., a detectable or therapeutic agent, at asecond end. The linker may be of sufficient length as to not interferewith incorporation into a nucleic acid sequence. The linker can be usedfor any useful purpose, such as to form mmRNA multimers (e.g., throughlinkage of two or more polynucleotides, primary constructs, or mmRNAmolecules) or mmRNA conjugates, as well as to administer a payload, asdescribed herein. Examples of chemical groups that can be incorporatedinto the linker include, but are not limited to, alkyl, alkenyl,alkynyl, amido, amino, ether, thioether, ester, alkylene,heteroalkylene, aryl, or heterocyclyl, each of which can be optionallysubstituted, as described herein. Examples of linkers include, but arenot limited to, unsaturated alkanes, polyethylene glycols (e.g.,ethylene or propylene glycol monomeric units, e.g., diethylene glycol,dipropylene glycol, triethylene glycol, tripropylene glycol,tetraethylene glycol, or tetraethylene glycol), and dextran polymers,Other examples include, but are not limited to, cleavable moietieswithin the linker, such as, for example, a disulfide bond (—S—S—) or anazo bond (—N═N—), which can be cleaved using a reducing agent orphotolysis. Non-limiting examples of a selectively cleavable bondinclude an amido bond can be cleaved for example by the use oftris(2-carboxyethyl)phosphine (TCEP), or other reducing agents, and/orphotolysis, as well as an ester bond can be cleaved for example byacidic or basic hydrolysis.

MicroRNA (miRNA) binding site: As used herein, a microRNA (miRNA)binding site represents a nucleotide location or region of a nucleicacid transcript to which at least the “seed” region of a miRNA binds.

Modified: As used herein “modified” refers to a changed state orstructure of a molecule of the invention. Molecules may be modified inmany ways including chemically, structurally, and functionally. In oneembodiment, the mRNA molecules of the present invention are modified bythe introduction of non-natural nucleosides and/or nucleotides, e.g., asit relates to the natural ribonucleotides A, U, G, and C. Noncanonicalnucleotides such as the cap structures are not considered “modified”although they differ from the chemical structure of the A, C, G, Uribonucleotides.

Mucus: As used herein, “mucus” refers to the natural substance that isviscous and comprises mucin glycoproteins.

Multipotent: As used herein, “multipotent” or “partially differentiatedcell” when referring to a cell refers to a cell that has a developmentalpotential to differentiate into cells of one or more germ layers, butnot all three germ layers.

Naturally occurring: As used herein, “naturally occurring” meansexisting in nature without artificial aid.

Non-human vertebrate: As used herein, a “non human vertebrate” includesall vertebrates except Homo sapiens, including wild and domesticatedspecies. Examples of non-human vertebrates include, but are not limitedto, mammals, such as alpaca, banteng, bison, camel, cat, cattle, deer,dog, donkey, gayal, goat, guinea pig, horse, llama, mule, pig, rabbit,reindeer, sheep water buffalo, and yak.

Off-target: As used herein, “off target” refers to any unintended effecton any one or more target, gene, or cellular transcript.

Oligopotent: As used herein, “oligopotent” when referring to a cellmeans to give rise to a more restricted subset of cell lineages thanmultipotent stem cells.

Open reading frame: As used herein, “open reading frame” or “ORF” refersto a sequence which does not contain a stop codon in a given readingframe.

Operably linked: As used herein, the phrase “operably linked” refers toa functional connection between two or more molecules, constructs,transcripts, entities, moieties or the like.

Optionally substituted: Herein a phrase of the form “optionallysubstituted X” (e.g., optionally substituted alkyl) is intended to beequivalent to “X, wherein X is optionally substituted” (e.g., “alkyl,wherein said alkyl is optionally substituted”). It is not intended tomean that the feature “X” (e.g. alkyl)per se is optional.

Peptide: As used herein, “peptide” is less than or equal to 50 aminoacids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 aminoacids long.

Paratope: As used herein, a “paratope” refers to the antigen-bindingsite of an antibody.

Patient: As used herein, “patient” refers to a subject who may seek orbe in need of treatment, requires treatment, is receiving treatment,will receive treatment, or a subject who is under care by a trainedprofessional for a particular disease or condition.

Pharmaceutically acceptable: The phrase “pharmaceutically acceptable” isemployed herein to refer to those compounds, materials, compositions,and/or dosage forms which are, within the scope of sound medicaljudgment, suitable for use in contact with the tissues of human beingsand animals without excessive toxicity, irritation, allergic response,or other problem or complication, commensurate with a reasonablebenefit/risk ratio.

Pharmaceutically acceptable excipients: The phrase “pharmaceuticallyacceptable excipient,” as used herein, refers any ingredient other thanthe compounds described herein (for example, a vehicle capable ofsuspending or dissolving the active compound) and having the propertiesof being substantially nontoxic and non-inflammatory in a patient.Excipients may include, for example: antiadherents, antioxidants,binders, coatings, compression aids, disintegrants, dyes (colors),emollients, emulsifiers, fillers (diluents), film formers or coatings,flavors, fragrances, glidants (flow enhancers), lubricants,preservatives, printing inks, sorbents, suspensing or dispersing agents,sweeteners, and waters of hydration. Exemplary excipients include, butare not limited to: butylated hydroxytoluene (BHT), calcium carbonate,calcium phosphate (dibasic), calcium stearate, croscarmellose,crosslinked polyvinyl pyrrolidone, citric acid, crospovidone, cysteine,ethylcellulose, gelatin, hydroxypropyl cellulose, hydroxypropylmethylcellulose, lactose, magnesium stearate, maltitol, mannitol,methionine, methylcellulose, methyl paraben, microcrystalline cellulose,polyethylene glycol, polyvinyl pyrrolidone, povidone, pregelatinizedstarch, propyl paraben, retinyl palmitate, shellac, silicon dioxide,sodium carboxymethyl cellulose, sodium citrate, sodium starch glycolate,sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide,vitamin A, vitamin E, vitamin C, and xylitol.

Pharmaceutically acceptable salts: The present disclosure also includespharmaceutically acceptable salts of the compounds described herein. Asused herein, “pharmaceutically acceptable salts” refers to derivativesof the disclosed compounds wherein the parent compound is modified byconverting an existing acid or base moiety to its salt form (e.g., byreacting the free base group with a suitable organic acid). Examples ofpharmaceutically acceptable salts include, but are not limited to,mineral or organic acid salts of basic residues such as amines; alkalior organic salts of acidic residues such as carboxylic acids; and thelike. Representative acid addition salts include acetate, adipate,alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate,borate, butyrate, camphorate, camphorsulfonate, citrate,cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate,fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate,hexanoate, hydrobromide, hydrochloride, hydroiodide,2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, laurylsulfate, malate, maleate, malonate, methanesulfonate,2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate,pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate,pivalate, propionate, stearate, succinate, sulfate, tartrate,thiocyanate, toluenesulfonate, undecanoate, valerate salts, and thelike. Representative alkali or alkaline earth metal salts includesodium, lithium, potassium, calcium, magnesium, and the like, as well asnontoxic ammonium, quaternary ammonium, and amine cations, including,but not limited to ammonium, tetramethylammonium, tetraethylammonium,methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine,and the like. The pharmaceutically acceptable salts of the presentdisclosure include the conventional non-toxic salts of the parentcompound formed, for example, from non-toxic inorganic or organic acids.The pharmaceutically acceptable salts of the present disclosure can besynthesized from the parent compound which contains a basic or acidicmoiety by conventional chemical methods. Generally, such salts can beprepared by reacting the free acid or base forms of these compounds witha stoichiometric amount of the appropriate base or acid in water or inan organic solvent, or in a mixture of the two; generally, nonaqueousmedia like ether, ethyl acetate, ethanol, isopropanol, or acetonitrileare preferred. Lists of suitable salts are found in Remington'sPharmaceutical Sciences, 17^(th) ed., Mack Publishing Company, Easton,Pa., 1985, p. 1418, Pharmaceutical Salts: Properties, Selection, andUse, P. H. Stahl and C. G. Wermuth (eds.), Wiley-VCH, 2008, and Berge etal., Journal of Pharmaceutical Science, 66, 1-19 (1977), each of whichis incorporated herein by reference in its entirety.

Pharmacokinetic: As used herein, “pharmacokinetic” refers to any one ormore properties of a molecule or compound as it relates to thedetermination of the fate of substances administered to a livingorganism. Pharmacokinetics is divided into several areas including theextent and rate of absorption, distribution, metabolism and excretion.This is commonly referred to as ADME where: (A) Absorption is theprocess of a substance entering the blood circulation; (D) Distributionis the dispersion or dissemination of substances throughout the fluidsand tissues of the body; (M) Metabolism (or Biotransformation) is theirreversible transformation of parent compounds into daughtermetabolites; and (E) Excretion (or Elimination) refers to theelimination of the substances from the body. In rare cases, some drugsirreversibly accumulate in body tissue.

Pharmaceutically acceptable solvate: The term “pharmaceuticallyacceptable solvate,” as used herein, means a compound of the inventionwherein molecules of a suitable solvent are incorporated in the crystallattice. A suitable solvent is physiologically tolerable at the dosageadministered. For example, solvates may be prepared by crystallization,recrystallization, or precipitation from a solution that includesorganic solvents, water, or a mixture thereof. Examples of suitablesolvents are ethanol, water (for example, mono-, di-, and tri-hydrates),N-methylpyrrolidinone (NMP), dimethyl sulfoxide (DMSO),N,N′-dimethylformamide (DMF), N,N′-dimethylacetamide (DMAC),1,3-dimethyl-2-imidazolidinone (DMEU),1,3-dimethyl-3,4,5,6-tetrahydro-2-(1H)-pyrimidinone (DMPU), acetonitrile(ACN), propylene glycol, ethyl acetate, benzyl alcohol, 2-pyrrolidone,benzyl benzoate, and the like. When water is the solvent, the solvate isreferred to as a “hydrate.”

Physicochemical: As used herein, “physicochemical” means of or relatingto a physical and/or chemical property.

Pluripotent: As used herein, “pluripotent” refers to a cell with thedevelopmental potential, under different conditions, to differentiate tocell types characteristic of all three germ layers.

Pluripotency: As used herein, “pluripotency” or “pluripotent state”refers to the developmental potential of a cell where the cell has theability to differentitate into all three embryonic germ layers(endoderm, mesoderm and ectoderm).

Preventing: As used herein, the term “preventing” refers to partially orcompletely delaying onset of an infection, disease, disorder and/orcondition; partially or completely delaying onset of one or moresymptoms, features, or clinical manifestations of a particularinfection, disease, disorder, and/or condition; partially or completelydelaying onset of one or more symptoms, features, or manifestations of aparticular infection, disease, disorder, and/or condition; partially orcompletely delaying progression from an infection, a particular disease,disorder and/or condition; and/or decreasing the risk of developingpathology associated with the infection, the disease, disorder, and/orcondition.

Prodrug: The present disclosure also includes prodrugs of the compoundsdescribed herein. As used herein, “prodrugs” refer to any substance,molecule or entity which is in a form predicate for that substance,molecule or entity to act as a therapeutic upon chemical or physicalalteration. Prodrugs may by covalently bonded or sequestered in some wayand which release or are converted into the active drug moiety prior to,upon or after administered to a mammalian subject. Prodrugs can beprepared by modifying functional groups present in the compounds in sucha way that the modifications are cleaved, either in routine manipulationor in vivo, to the parent compounds. Prodrugs include compounds whereinhydroxyl, amino, sulfhydryl, or carboxyl groups are bonded to any groupthat, when administered to a mammalian subject, cleaves to form a freehydroxyl, amino, sulfhydryl, or carboxyl group respectively. Preparationand use of prodrugs is discussed in T. Higuchi and V. Stella, “Pro-drugsas Novel Delivery Systems,” Vol. 14 of the A.C.S. Symposium Series, andin Bioreversible Carriers in Drug Design, ed. Edward B. Roche, AmericanPharmaceutical Association and Pergamon Press, 1987, both of which arehereby incorporated by reference in their entirety.

Proliferate: As used herein, the term “proliferate” means to grow,expand or increase or cause to grow, expand or increase rapidly.“Proliferative” means having the ability to proliferate.“Anti-proliferative” means having properties counter to or inapposite toproliferative properties.

Progenitor cell: As used herein, the term “progenitor cell” refers tocells that have greater developmental potential relative to a cell whichit can give rise to by differentiation.

Protein cleavage site: As used herein, “protein cleavage site” refers toa site where controlled cleavage of the amino acid chain can beaccomplished by chemical, enzymatic or photochemical means.

Protein cleavage signal: As used herein “protein cleavage signal” refersto at least one amino acid that flags or marks a polypeptide forcleavage.

Protein of interest: As used herein, the terms “proteins of interest” or“desired proteins” include those provided herein and fragments, mutants,variants, and alterations thereof.

Proximal: As used herein, the term “proximal” means situated nearer tothe center or to a point or region of interest.

Purified: As used herein, “purify,” “purified,” “purification” means tomake substantially pure or clear from unwanted components, materialdefilement, admixture or imperfection.

Repeated transfection: As used herein, the term “repeated transfection”refers to transfection of the same cell culture with a polynucleotide,primary construct or mmRNA a plurality of times. The cell culture can betransfected at least twice, at least 3 times, at least 4 times, at least5 times, at least 6 times, at least 7 times, at least 8 times, at least9 times, at least 10 times, at least 11 times, at least 12 times, atleast 13 times, at least 14 times, at least 15 times, at least 16 times,at least 17 times at least 18 times, at least 19 times, at least 20times, at least 25 times, at least 30 times, at least 35 times, at least40 times, at least 45 times, at least 50 times or more.

Reprogramming: As used herein, “reprogramming” refers to a process thatreverses the developmental potential of a cell or population of cells.

Reprogramming factor: As used herein, the term “reprogramming factor”refers to a developmental potential altering factor such as a protein,RNA or small molecule, the expression of which contributes to thereprogramming of a cell to a less differentiated or undifferentiatedstate.

Sample: As used herein, the term “sample” or “biological sample” refersto a subset of its tissues, cells or component parts (e.g. body fluids,including but not limited to blood, mucus, lymphatic fluid, synovialfluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic cord blood,urine, vaginal fluid and semen). A sample further may include ahomogenate, lysate or extract prepared from a whole organism or a subsetof its tissues, cells or component parts, or a fraction or portionthereof, including but not limited to, for example, plasma, serum,spinal fluid, lymph fluid, the external sections of the skin,respiratory, intestinal, and genitourinary tracts, tears, saliva, milk,blood cells, tumors, organs. A sample further refers to a medium, suchas a nutrient broth or gel, which may contain cellular components, suchas proteins or nucleic acid molecule.

Signal Sequences: As used herein, the phrase “signal sequences” refersto a sequence which can direct the transport or localization of aprotein.

Single unit dose: As used herein, a “single unit dose” is a dose of anytherapeutic administed in one dose/at one time/single route/single pointof contact, i.e., single administration event.

Similarity: As used herein, the term “similarity” refers to the overallrelatedness between polymeric molecules, e.g. between polynucleotidemolecules (e.g. DNA molecules and/or RNA molecules) and/or betweenpolypeptide molecules. Calculation of percent similarity of polymericmolecules to one another can be performed in the same manner as acalculation of percent identity, except that calculation of percentsimilarity takes into account conservative substitutions as isunderstood in the art.

Somatic cell: As used herein, “somatic cells” refers to any cell otherthan a germ cell, a cell present in or obtained from a pre-implantationembryo, or a cell resulting from proliferation of such a cell in vitro.

Somatic stem cell: As used herein, a “somatic stem cell” refers to anypluripotent or multipotent stem cell derived from non-embryonic tissueincluding fetal, juvenile and adult tissue.

Somatic pluripotent cell: As used herein, a “somatic pluripotent cell”refers to a somatic cell that has had its developmental potentialaltered to that of a pluripotent state.

Split dose: As used herein, a “split dose” is the division of singleunit dose or total daily dose into two or more doses.

Stable: As used herein “stable” refers to a compound that issufficiently robust to survive isolation to a useful degree of purityfrom a reaction mixture, and preferably capable of formulation into anefficacious therapeutic agent.

Stabilized: As used herein, the term “stabilize”, “stabilized,”“stabilized region” means to make or become stable.

Stem cell: As used herein, the term “stem cell” refers to a cell in anundifferentiated or partially differentiated state that has the propertyof self-renewal and ahs the developmental potential to differentiateinto multiple cell types, without a specific developmental potential. Astem cell may be able capable of proliferation and giving rise to moresuch stem cells while maintaining its developmental potential.

Subject: As used herein, the term “subject” or “patient” refers to anyorganism to which a composition in accordance with the invention may beadministered, e.g., for experimental, diagnostic, prophylactic, and/ortherapeutic purposes. Typical subjects include animals (e.g., mammalssuch as mice, rats, rabbits, non-human primates, and humans) and/orplants.

Substantially: As used herein, the term “substantially” refers to thequalitative condition of exhibiting total or near-total extent or degreeof a characteristic or property of interest. One of ordinary skill inthe biological arts will understand that biological and chemicalphenomena rarely, if ever, go to completion and/or proceed tocompleteness or achieve or avoid an absolute result. The term“substantially” is therefore used herein to capture the potential lackof completeness inherent in many biological and chemical phenomena.

Substantially equal: As used herein as it relates to time differencesbetween doses, the term means plus/minus 2%.

Substantially simultaneously: As used herein and as it relates toplurality of doses, the term means within 2 seconds.

Suffering from: An individual who is “suffering from” a disease,disorder, and/or condition has been diagnosed with or displays one ormore symptoms of a disease, disorder, and/or condition.

Susceptible to: An individual who is “susceptible to” a disease,disorder, and/or condition has not been diagnosed with and/or may notexhibit symptoms of the disease, disorder, and/or condition but harborsa propensity to develop a disease or its symptoms. In some embodiments,an individual who is susceptible to a disease, disorder, and/orcondition (for example, cancer) may be characterized by one or more ofthe following: (1) a genetic mutation associated with development of thedisease, disorder, and/or condition; (2) a genetic polymorphismassociated with development of the disease, disorder, and/or condition;(3) increased and/or decreased expression and/or activity of a proteinand/or nucleic acid associated with the disease, disorder, and/orcondition; (4) habits and/or lifestyles associated with development ofthe disease, disorder, and/or condition; (5) a family history of thedisease, disorder, and/or condition; and (6) exposure to and/orinfection with a microbe associated with development of the disease,disorder, and/or condition. In some embodiments, an individual who issusceptible to a disease, disorder, and/or condition will develop thedisease, disorder, and/or condition. In some embodiments, an individualwho is susceptible to a disease, disorder, and/or condition will notdevelop the disease, disorder, and/or condition.

Sustained release: As used herein, the term “sustained release” refersto a pharmaceutical composition or compound release profile thatconforms to a release rate over a specific period of time.

Synthetic: The term “synthetic” means produced, prepared, and/ormanufactured by the hand of man. Synthesis of polynucleotides orpolypeptides or other molecules of the present invention may be chemicalor enzymatic.

Targeted Cells: As used herein, “targeted cells” refers to any one ormore cells of interest. The cells may be found in vitro, in vivo, insitu or in the tissue or organ of an organism. The organism may be ananimal, preferably a mammal, more preferably a human and most preferablya patient.

Therapeutic Agent: The term “therapeutic agent” refers to any agentthat, when administered to a subject, has a therapeutic, diagnostic,and/or prophylactic effect and/or elicits a desired biological and/orpharmacological effect.

Therapeutically effective amount: As used herein, the term“therapeutically effective amount” means an amount of an agent to bedelivered (e.g., nucleic acid, drug, therapeutic agent, diagnosticagent, prophylactic agent, etc.) that is sufficient, when administeredto a subject suffering from or susceptible to an infection, disease,disorder, and/or condition, to treat, improve symptoms of, diagnose,prevent, and/or delay the onset of the infection, disease, disorder,and/or condition.

Therapeutically effective outcome: As used herein, the term“therapeutically effective outcome” means an outcome that is sufficientin a subject suffering from or susceptible to an infection, disease,disorder, and/or condition, to treat, improve symptoms of, diagnose,prevent, and/or delay the onset of the infection, disease, disorder,and/or condition.

Total daily dose: As used herein, a “total daily dose” is an amountgiven or prescribed in 24 hr period. It may be administered as a singleunit dose.

Totipotency: As used herein, “totipotency” refers to a cell with adevelopmental potential to make all of the cells found in the adult bodyas well as the extra-embryonic tissues, including the placenta.

Transcription factor: As used herein, the term “transcription factor”refers to a DNA-binding protein that regulates transcription of DNA intoRNA, for example, by activation or repression of transcription. Sometranscription factors effect regulation of transcription alone, whileothers act in concert with other proteins. Some transcription factor canboth activate and repress transcription under certain conditions. Ingeneral, transcription factors bind a specific target sequence orsequences highly similar to a specific consensus sequence in aregulatory region of a target gene. Transcription factors may regulatetranscription of a target gene alone or in a complex with othermolecules.

Transcription: As used herein, the term “transcription” refers tomethods to introduce exogenous nucleic acids into a cell. Methods oftransfection include, but are not limited to, chemical methods, physicaltreatments and cationic lipids or mixtures.

Transdifferentiation: As used herein, “transdifferentiation” refers tothe capacity of differentiated cells of one type to lose identifyingcharacteristics and to change their phenotype to that of other fullydifferentiated cells.

Treating: As used herein, the term “treating” refers to partially orcompletely alleviating, ameliorating, improving, relieving, delayingonset of, inhibiting progression of, reducing severity of, and/orreducing incidence of one or more symptoms or features of a particularinfection, disease, disorder, and/or condition. For example, “treating”cancer may refer to inhibiting survival, growth, and/or spread of atumor. Treatment may be administered to a subject who does not exhibitsigns of a disease, disorder, and/or condition and/or to a subject whoexhibits only early signs of a disease, disorder, and/or condition forthe purpose of decreasing the risk of developing pathology associatedwith the disease, disorder, and/or condition.

Unmodified: As used herein, “unmodified” refers to any substance,compound or molecule prior to being changed in any way. Unmodified may,but does not always, refer to the wild type or native form of abiomolecule. Molecules may undergo a series of modifications wherebyeach modified molecule may serve as the “unmodified” starting moleculefor a subsequent modification.

EQUIVALENTS AND SCOPE

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments in accordance with the invention described herein. The scopeof the present invention is not intended to be limited to the aboveDescription, but rather is as set forth in the appended claims.

In the claims, articles such as “a,” “an,” and “the” may mean one ormore than one unless indicated to the contrary or otherwise evident fromthe context. Claims or descriptions that include “or” between one ormore members of a group are considered satisfied if one, more than one,or all of the group members are present in, employed in, or otherwiserelevant to a given product or process unless indicated to the contraryor otherwise evident from the context. The invention includesembodiments in which exactly one member of the group is present in,employed in, or otherwise relevant to a given product or process. Theinvention includes embodiments in which more than one, or all of thegroup members are present in, employed in, or otherwise relevant to agiven product or process.

It is also noted that the term “comprising” is intended to be open andpermits but does not require the inclusion of additional elements orsteps. When the term “comprising” is used herein, the term “consistingof” is thus also encompassed and disclosed.

Where ranges are given, endpoints are included. Furthermore, it is to beunderstood that unless otherwise indicated or otherwise evident from thecontext and understanding of one of ordinary skill in the art, valuesthat are expressed as ranges can assume any specific value or subrangewithin the stated ranges in different embodiments of the invention, tothe tenth of the unit of the lower limit of the range, unless thecontext clearly dictates otherwise.

In addition, it is to be understood that any particular embodiment ofthe present invention that falls within the prior art may be explicitlyexcluded from any one or more of the claims. Since such embodiments aredeemed to be known to one of ordinary skill in the art, they may beexcluded even if the exclusion is not set forth explicitly herein. Anyparticular embodiment of the compositions of the invention (e.g., anynucleic acid or protein encoded thereby; any method of production; anymethod of use; etc.) can be excluded from any one or more claims, forany reason, whether or not related to the existence of prior art.

All cited sources, for example, references, publications, databases,database entries, and art cited herein, are incorporated into thisapplication by reference, even if not expressly stated in the citation.In case of conflicting statements of a cited source and the instantapplication, the statement in the instant application shall control.

Section and table headings are not intended to be limiting.

EXAMPLES Example 1. Modified mRNA Production

Modified mRNAs (mmRNA) according to the invention may be made usingstandard laboratory methods and materials. The open reading frame (ORF)of the gene of interest may be flanked by a 5′ untranslated region (UTR)which may contain a strong Kozak translational initiation signal and/oran alpha-globin 3′ UTR which may include an oligo(dT) sequence fortemplated addition of a poly-A tail. The modified mRNAs may be modifiedto reduce the cellular innate immune response. The modifications toreduce the cellular response may include pseudouridine (ψ) and5-methyl-cytidine (5meC, 5mc or m⁵C). (See, Kariko K et al. Immunity23:165-75 (2005), Kariko K et al. Mol Ther 16:1833-40 (2008), Anderson BR et al. NAR (2010); each of which is herein incorporated by referencein their entirety).

The ORF may also include various upstream or downstream additions (suchas, but not limited to, β-globin, tags, etc.) may be ordered from anoptimization service such as, but limited to, DNA2.0 (Menlo Park,Calif.) and may contain multiple cloning sites which may have XbaIrecognition. Upon receipt of the construct, it may be reconstituted andtransformed into chemically competent E. coli.

For the present invention, NEB DH5-alpha Competent E. coli are used.Transformations are performed according to NEB instructions using 100 ngof plasmid. The protocol is as follows:

-   -   1. Thaw a tube of NEB 5-alpha Competent E. coli cells on ice for        10 minutes.    -   2. Add 1-5 μl containing 1 pg-100 ng of plasmid DNA to the cell        mixture. Carefully flick the tube 4-5 times to mix cells and        DNA. Do not vortex.    -   3. Place the mixture on ice for 30 minutes. Do not mix.    -   4. Heat shock at 42° C. for exactly 30 seconds. Do not mix.    -   5. Place on ice for 5 minutes. Do not mix.    -   6. Pipette 950 μl of room temperature SOC into the mixture.    -   7. Place at 37° C. for 60 minutes. Shake vigorously (250 rpm) or        rotate.    -   8. Warm selection plates to 37° C.    -   9. Mix the cells thoroughly by flicking the tube and inverting.

Alternatively, incubate at 30° C. for 24-36 hours or 25° C. for 48hours.

A single colony is then used to inoculate 5 ml of LB growth media usingthe appropriate antibiotic and then allowed to grow (250 RPM, 37° C.)for 5 hours. This is then used to inoculate a 200 ml culture medium andallowed to grow overnight under the same conditions.

To isolate the plasmid (up to 850 μg), a maxi prep is performed usingthe Invitrogen PURELINK™ HiPure Maxiprep Kit (Carlsbad, Calif.),following the manufacturer's instructions.

In order to generate cDNA for In Vitro Transcription (IVT), the plasmidis first linearized using a restriction enzyme such as XbaI. A typicalrestriction digest with XbaI will comprise the following: Plasmid 1.0μg; 10× Buffer 1.0 μl; XbaI 1.5 μl; dH₂O up to 10 μl; incubated at 37°C. for 1 hr. If performing at lab scale (<5 μg), the reaction is cleanedup using Invitrogen's PURELINK™ PCR Micro Kit (Carlsbad, Calif.) permanufacturer's instructions. Larger scale purifications may need to bedone with a product that has a larger load capacity such as Invitrogen'sstandard PURELINK™ PCR Kit (Carlsbad, Calif.). Following the cleanup,the linearized vector is quantified using the NanoDrop and analyzed toconfirm linearization using agarose gel electrophoresis.

As a non-limiting example, G-CSF may represent the polypeptide ofinterest. Sequences used in the steps outlined in Examples 1-5 are shownin Table 3. It should be noted that the start codon (ATG) has beenunderlined in each sequence of Table 3.

TABLE 3 G-CSF Sequences SEQ ID NO Description 27 cDNAsequence:ATGGCTGGACCTGCCACCCAGAGCCCCATGAAGCTGATGGCCCTGCAGCTGCTGCTGTGGCACAGTGCACTCTGGACAGTGCAGGAAGCCACCCCCCTGGGCCCTGCCAGCTCCCTGCCCCAGAGCTTCCTGCTCAAGTGCTTAGAGCAAGTGAGGAAGATCCAGGGCGATGGCGCAGCGCTCCAGGAGAAGCTGTGTGCCACCTACAAGCTGTGCCACCCCGAGGAGCTGGTGCTGCTCGGACACTCTCTGGGCATCCCCTGGGCTCCCCTGAGCAGCTGCCCCAGCCAGGCCCTGCAGCTGGCAGGCTGCTTGAGCCAACTCCATAGCGGCCTTTTCCTCTACCAGGGGCTCCTGCAGGCCCTGGAAGGGATCTCCCCCGAGTTGGGTCCCACCTTGGACACACTGCAGCTGGACGTCGCCGACTTTGCCACCACCATCTGGCAGCAGATGGAAGAACTGGGAATGGCCCCTGCCCTGCAGCCCACCCAGGGTGCCATGCCGGCCTTCGCCTCTGCTTTCCAGCGCCGGGCAGGAGGGGTCCTGGTTGCCTCCCATCTGCAGAGCTTCCTGGAGGTGTCGTACCGCGTTCTACGCCACCTTGCCCAGCCCTGA 28cDNA having T7 polymerase site. AfeI and Xba restriction site:TAATACGACTCACTATA GGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGCTGGACCTGCCACCCAGAGCCCCATGAAGCTGATGGCCCTGCAGCTGCTGCTGTGGCACAGTGCACTCTGGACAGTGCAGGAAGCCACCCCCCTGGGCCCTGCCAGCTCCCTGCCCCAGAGCTTCCTGCTCAAGTGCTTAGAGCAAGTGAGGAAGATCCAGGGCGATGGCGCAGCGCTCCAGGAGAAGCTGTGTGCCACCTACAAGCTGTGCCACCCCGAGGAGCTGGTGCTGCTCGGACACTCTCTGGGCATCCCCTGGGCTCCCCTGAGCAGCTGCCCCAGCCAGGCCCTGCAGCTGGCAGGCTGCTTGAGCCAACTCCATAGCGGCCTTTTCCTCTACCAGGGGCTCCTGCAGGCCCTGGAAGGGATCTCCCCCGAGTTGGGTCCCACCTTGGACACACTGCAGCTGGACGTCGCCGACTTTGCCACCACCATCTGGCAGCAGATGGAAGAACTGGGAATGGCCCCTGCCCTGCAGCCCACCCAGGGTGCCATGCCGGCCTTCGCCTCTGCTTTCCAGCGCCGGGCAGGAGGGGTCCTGGTTGCCTCCCATCTGCAGAGCTTCCTGGAGGTGTCGTACCGCGTTCTACGCCACCTTGCCCAGCCCTGAAGCGCTGCCTTCTGCGGGGCTTGCCTTCTGGCCATGCCCTTCTTCTCTCCCTTGCACCTGTACCTCTTGGTCTTTGAATAAAGCCTGAGTAGGAAGGCG GCCGCTCGAGCATGCATCTAGA29 Optimized sequence; containing T7 polymerase site. AfeI and Xbarestriction site TAATACGACTCACTATAGGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGCCGGTCCCGCGACCCAAAGCCCCATGAAACTTATGGCCCTGCAGTTGCTGCTTTGGCACTCGGCCCTCTGGACAGTCCAAGAAGCGACTCCTCTCGGACCTGCCTCATCGTTGCCGCAGTCATTCCTTTTGAAGTGTCTGGAGCAGGTGCGAAAGATTCAGGGCGATGGAGCCGCACTCCAAGAGAAGCTCTGCGCGACATACAAACTTTGCCATCCCGAGGAGCTCGTACTGCTCGGGCACAGCTTGGGGATTCCCTGGGCTCCTCTCTCGTCCTGTCCGTCGCAGGCTTTGCAGTTGGCAGGGTGCCTTTCCCAGCTCCACTCCGGTTTGTTCTTGTATCAGGGACTGCTGCAAGCCCTTGAGGGAATCTCGCCAGAATTGGGCCCGACGCTGGACACGTTGCAGCTCGACGTGGCGGATTTCGCAACAACCATCTGGCAGCAGATGGAGGAACTGGGGATGGCACCCGCGCTGCAGCCCACGCAGGGGGCAATGCCGGCCTTTGCGTCCGCGTTTCAGCGCAGGGCGGGTGGAGTCCTCGTAGCGAGCCACCTTCAATCATTTTTGGAAGTCTCGTACCGGGTGCTGAGACATCTTGCGCAGCCGTGAAGCGCTGCCTTCTGCGGGGCTTGCCTTCTGGCCATGCCCTTCTTCTCTCCCTTGCACCTGTACCTCTTGGTCTTTGAATAAAGCCTGAGTAGGAAGGCG GCCGCTCGAGCATGCATCTAGA30 mRNA sequence (transcribed)GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUGGCCGGUCCCGCGACCCAAAGCCCCAUGAAACUUAUGGCCCUGCAGUUGCUGCUUUGGCACUCGGCCCUCUGGACAGUCCAAGAAGCGACUCCUCUCGGACCUGCCUCAUCGUUGCCGCAGUCAUUCCUUUUGAAGUGUCUGGAGCAGGUGCGAAAGAUUCAGGGCGAUGGAGCCGCACUCCAAGAGAAGCUCUGCGCGACAUACAAACUUUGCCAUCCCGAGGAGCUCGUACUGCUCGGGCACAGCUUGGGGAUUCCCUGGGCUCCUCUCUCGUCCUGUCCGUCGCAGGCUUUGCAGUUGGCAGGGUGCCUUUCCCAGCUCCACUCCGGUUUGUUCUUGUAUCAGGGACUGCUGCAAGCCCUUGAGGGAAUCUCGCCAGAAUUGGGCCCGACGCUGGACACGUUGCAGCUCGACGUGGCGGAUUUCGCAACAACCAUCUGGCAGCAGAUGGAGGAACUGGGGAUGGCACCCGCGCUGCAGCCCACGCAGGGGGCAAUGCCGGCCUUUGCGUCCGCGUUUCAGCGCAGGGCGGGUGGAGUCCUCGUAGCGAGCCACCUUCAAUCAUUUUUGGAAGUCUCGUACCGGGUGCUGAGACAUCUUGCGCAGCCGUGAAGCGCUGCCUUCUGCGGGGCUUGCCUUCUGGCCAUGCCCUUCUUCUCUCCCUUGCACCUGUACCUCUUGGUCUUUGAAUAAAGCCUGAGUAGGAA G

Example 2: PCR for cDNA Production

PCR procedures for the preparation of cDNA are performed using 2×KAPAHIFI™ HotStart ReadyMix by Kapa Biosystems (Woburn, Mass.). This systemincludes 2×KAPA ReadyMix12.5 μl; Forward Primer (10 uM) 0.75 μl; ReversePrimer (10 uM) 0.75 μl; Template cDNA 100 ng; and dH₂O diluted to 25.0μl. The reaction conditions are at 95° C. for 5 min. and 25 cycles of98° C. for 20 sec, then 58° C. for 15 sec, then 72° C. for 45 sec, then72° C. for 5 min. then 4° C. to termination.

The reverse primer of the instant invention incorporates a poly-T₁₂₀ fora poly-A₁₂₀ in the mRNA. Other reverse primers with longer or shorterpoly(T) tracts can be used to adjust the length of the poly(A) tail inthe mRNA.

The reaction is cleaned up using Invitrogen's PURELINK™ PCR Micro Kit(Carlsbad, Calif.) per manufacturer's instructions (up to 5 μg). Largerreactions will require a cleanup using a product with a larger capacity.Following the cleanup, the cDNA is quantified using the NanoDrop andanalyzed by agarose gel electrophoresis to confirm the cDNA is theexpected size. The cDNA is then submitted for sequencing analysis beforeproceeding to the in vitro transcription reaction.

Example 3. In Vitro Transcription (IVT)

The in vitro transcription reaction generates mRNA containing modifiednucleotides or modified RNA. The input nucleotide triphosphate (NTP) mixis made in-house using natural and un-natural NTPs.

A typical in vitro transcription reaction includes the following:

Template cDNA 1.0 μg 10x transcription buffer (400 mM Tris-HCl 2.0 μl pH8.0, 190 mM MgCl₂, 50 mM DTT, 10 mM Spermidine) Custom NTPs (25 mM each)7.2 μl RNase Inhibitor 20 U T7 RNA polymerase 3000 U dH₂0 Up to 20.0 μlIncubation at 37° C. for 3 hr-5 hrs.

The crude IVT mix may be stored at 4° C. overnight for cleanup the nextday. 1 U of RNase-free DNase is then used to digest the originaltemplate. After 15 minutes of incubation at 37° C., the mRNA is purifiedusing Ambion's MEGACLEAR™ Kit (Austin, Tex.) following themanufacturer's instructions. This kit can purify up to 500 μg of RNA.Following the cleanup, the RNA is quantified using the NanoDrop andanalyzed by agarose gel electrophoresis to confirm the RNA is the propersize and that no degradation of the RNA has occurred.

Example 4. Enzymatic Capping of mRNA

Capping of the mRNA is performed as follows where the mixture includes:IVT RNA 60 μg-180 μg and dH₂O up to 72 μl. The mixture is incubated at65° C. for 5 minutes to denature RNA, and then is transferredimmediately to ice.

The protocol then involves the mixing of 10× Capping Buffer (0.5 MTris-HCl (pH 8.0), 60 mM KCl, 12.5 mM MgCl₂) (10.0 μl); 20 mM GTP (5.0μl); 20 mM S-Adenosyl Methionine (2.5 μl); RNase Inhibitor (100 U);2′-O-Methyltransferase (400U); Vaccinia capping enzyme (Guanylyltransferase) (40 U); dH₂O (Up to 28 μl); and incubation at 37° C. for 30minutes for 60 μs RNA or up to 2 hours for 180 μs of RNA.

The mRNA is then purified using Ambion's MEGACLEAR™ Kit (Austin, Tex.)following the manufacturer's instructions. Following the cleanup, theRNA is quantified using the NANODROP™ (ThermoFisher, Waltham, Mass.) andanalyzed by agarose gel electrophoresis to confirm the RNA is the propersize and that no degradation of the RNA has occurred. The RNA productmay also be sequenced by running a reverse-transcription-PCR to generatethe cDNA for sequencing.

Example 5. PolyA Tailing Reaction

Without a poly-T in the cDNA, a poly-A tailing reaction must beperformed before cleaning the final product. This is done by mixingCapped IVT RNA (100 μl); RNase Inhibitor (20 U); 10× Tailing Buffer (0.5M Tris-HCl (pH 8.0), 2.5 M NaCl, 100 mM MgCl₂)(12.0 μl); 20 mM ATP (6.0μl); Poly-A Polymerase (20 U); dH₂O up to 123.5 μl and incubation at 37°C. for 30 min. If the poly-A tail is already in the transcript, then thetailing reaction may be skipped and proceed directly to cleanup withAmbion's MEGACLEAR™ kit (Austin, Tex.) (up to 500 μg). Poly-A Polymeraseis preferably a recombinant enzyme expressed in yeast.

For studies performed and described herein, the poly-A tail is encodedin the IVT template to comprise 160 nucleotides in length. However, itshould be understood that the processivity or integrity of the polyAtailing reaction may not always result in exactly 160 nucleotides. HencepolyA tails of approximately 160 nucleotides, e.g, about 150-165, 155,156, 157, 158, 159, 160, 161, 162, 163, 164 or 165 are within the scopeof the invention.

Example 6. Natural 5′ Caps and 5′ Cap Analogues

5′-capping of modified RNA may be completed concomitantly during the invitro-transcription reaction using the following chemical RNA capanalogs to generate the 5′-guanosine cap structure according tomanufacturer protocols: 3′-O-Me-m7G(5)ppp(5′) G [the ARCAcap];G(5)ppp(5′)A; G(5′)ppp(5′)G; m7G(5′)ppp(5′)A; m7G(5′)ppp(5′)G (NewEngland BioLabs, Ipswich, Mass.). 5′-capping of modified RNA may becompleted post-transcriptionally using a Vaccinia Virus Capping Enzymeto generate the “Cap 0” structure: m7G(5′)ppp(5′)G (New England BioLabs,Ipswich, Mass.). Cap 1 structure may be generated using both VacciniaVirus Capping Enzyme and a 2′-O methyl-transferase to generate:m7G(5′)ppp(5′)G-2′-O-methyl. Cap 2 structure may be generated from theCap 1 structure followed by the 2′-O-methylation of the5′-antepenultimate nucleotide using a 2′-O methyl-transferase. Cap 3structure may be generated from the Cap 2 structure followed by the2′-O-methylation of the 5′-preantepenultimate nucleotide using a 2′-Omethyl-transferase. Enzymes are preferably derived from a recombinantsource.

When transfected into mammalian cells, the modified mRNAs have astability of between 12-18 hours or more than 18 hours, e.g., 24, 36,48, 60, 72 or greater than 72 hours.

Example 7. Capping

A. Protein Expression Assay

Synthetic mRNAs encoding human G-CSF (mRNA sequence shown in SEQ ID NO:30 with a polyA tail approximately 160 nucleotides in length not shownin sequence) containing the ARCA (3′ O-Me-m7G(5′)ppp(5′)G) cap analog orthe Cap1 structure can be transfected into human primary keratinocytesat equal concentrations. 6, 12, 24 and 36 hours post-transfection theamount of G-CSF secreted into the culture medium can be assayed byELISA. Synthetic mRNAs that secrete higher levels of G-CSF into themedium would correspond to a synthetic mRNA with a highertranslationally-competent Cap structure.

B. Purity Analysis Synthesis

Synthetic mRNAs encoding human G-CSF (mRNA sequence shown in SEQ ID NO:30 with a polyA tail approximately 160 nucleotides in length not shownin sequence) containing the ARCA cap analog or the Cap1 structure crudesynthesis products can be compared for purity using denaturingAgarose-Urea gel electrophoresis or HPLC analysis. Synthetic mRNAs witha single, consolidated band by electrophoresis correspond to the higherpurity product compared to a synthetic mRNA with multiple bands orstreaking bands. Synthetic mRNAs with a single HPLC peak would alsocorrespond to a higher purity product. The capping reaction with ahigher efficiency would provide a more pure mRNA population.

C. Cytokine Analysis

Synthetic mRNAs encoding human G-CSF (mRNA sequence shown in SEQ ID NO:30; with a polyA tail approximately 160 nucleotides in length not shownin sequence) containing the ARCA cap analog or the Cap1 structure can betransfected into human primary keratinocytes at multiple concentrations.6, 12, 24 and 36 hours post-transfection the amount of pro-inflammatorycytokines such as TNF-alpha and IFN-beta secreted into the culturemedium can be assayed by ELISA. Synthetic mRNAs that secrete higherlevels of pro-inflammatory cytokines into the medium would correspond toa synthetic mRNA containing an immune-activating cap structure.

D. Capping Reaction Efficiency

Synthetic mRNAs encoding human G-CSF (mRNA shown in SEQ ID NO: 30 with apolyA tail approximately 160 nucleotides in length not shown insequence) containing the ARCA cap analog or the Cap1 structure can beanalyzed for capping reaction efficiency by LC-MS after capped mRNAnuclease treatment. Nuclease treatment of capped mRNAs would yield amixture of free nucleotides and the capped 5′-5-triphosphate capstructure detectable by LC-MS. The amount of capped product on the LC-MSspectra can be expressed as a percent of total mRNA from the reactionand would correspond to capping reaction efficiency. The cap structurewith higher capping reaction efficiency would have a higher amount ofcapped product by LC-MS.

Example 8. Agarose Gel Electrophoresis of Modified RNA or RT PCRProducts

Individual modified RNAs (200-400 ng in a 20 μl volume) or reversetranscribed PCR products (200-400 ng) are loaded into a well on anon-denaturing 1.2% Agarose E-Gel (Invitrogen, Carlsbad, Calif.) and runfor 12-15 minutes according to the manufacturer protocol.

Example 9. Nanodrop Modified RNA Quantification and UV Spectral Data

Modified RNAs in TE buffer (1 μl) are used for Nanodrop UV absorbancereadings to quantitate the yield of each modified RNA from an in vitrotranscription reaction.

Example 10. Method of Screening for Protein Expression

A. Electrospray Ionization

A biological sample which may contain proteins encoded by modified RNAadministered to the subject is prepared and analyzed according to themanufacturer protocol for electrospray ionization (ESI) using 1, 2, 3 or4 mass analyzers. A biologic sample may also be analyzed using a tandemESI mass spectrometry system.

Patterns of protein fragments, or whole proteins, are compared to knowncontrols for a given protein and identity is determined by comparison.

B. Matrix-Assisted Laser Desorption/Ionization

A biological sample which may contain proteins encoded by modified RNAadministered to the subject is prepared and analyzed according to themanufacturer protocol for matrix-assisted laser desorption/ionization(MALDI).

Patterns of protein fragments, or whole proteins, are compared to knowncontrols for a given protein and identity is determined by comparison.

C. Liquid Chromatography-Mass Spectrometry-Mass Spectrometry

A biological sample, which may contain proteins encoded by modified RNA,may be treated with a trypsin enzyme to digest the proteins containedwithin. The resulting peptides are analyzed by liquidchromatography-mass spectrometry-mass spectrometry (LC/MS/MS). Thepeptides are fragmented in the mass spectrometer to yield diagnosticpatterns that can be matched to protein sequence databases via computeralgorithms. The digested sample may be diluted to achieve 1 ng or lessstarting material for a given protein. Biological samples containing asimple buffer background (e.g. water or volatile salts) are amenable todirect in-solution digest; more complex backgrounds (e.g. detergent,non-volatile salts, glycerol) require an additional clean-up step tofacilitate the sample analysis.

Patterns of protein fragments, or whole proteins, are compared to knowncontrols for a given protein and identity is determined by comparison.

Example 11. LDL-R1 Mutant mRNA Sequences

Sequences encoding one or more LDL-R proteins which are deficient inPCSK9 binding are given in Table 4. The start site of the RNA isunderlined “AUG” and the 5′ UTR as well as the 3′UTR are bolded.

TABLE 4 LDL-R Sequences SEQ ID Description Sequence NO LDLR1_D331EGGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAG  7 mRNA AGCCACCAUGGGUCCGUGGGGCUGGAAGCUUAGAUGGACAGUCGCGCUCCUCCUUGCAGCAGCAGGAACUGCGGUCGGAGAUCGAUGCGAGCGCAACGAGUUCCAAUGCCAAGAUGGGAAGUGUAUUUCGUACAAGUGGGUCUGCGAUGGAUCAGCGGAAUGUCAGGACGGAAGCGAUGAGAGCCAAGAAACAUGCCUCUCAGUGACAUGCAAGUCGGGAGACUUCUCGUGCGGAGGACGCGUAAACAGAUGUAUUCCACAGUUUUGGCGCUGCGAUGGUCAGGUGGACUGCGACAACGGUUCAGAUGAACAGGGAUGUCCUCCGAAAACGUGCUCACAAGACGAGUUUCGCUGCCAUGAUGGAAAGUGCAUUUCGCGGCAGUUCGUAUGUGAUUCGGAUCGGGACUGUCUGGACGGCUCGGACGAAGCGUCAUGCCCGGUACUUACUUGCGGGCCAGCCUCAUUCCAAUGCAACAGCUCAACGUGCAUUCCCCAGCUGUGGGCCUGUGACAAUGAUCCUGAUUGUGAGGACGGUAGCGACGAGUGGCCGCAGAGAUGUAGGGGUUUGUACGUAUUCCAAGGAGACUCAAGCCCCUGUUCCGCCUUUGAGUUUCACUGCCUGUCGGGUGAAUGCAUCCACUCCAGCUGGCGAUGUGAUGGUGGGCCCGACUGCAAAGAUAAGAGCGACGAGGAGAAUUGCGCGGUCGCGACGUGCAGACCCGAUGAGUUCCAGUGCUCCGAUGGAAACUGCAUCCACGGGAGCCGGCAGUGUGAUCGCGAGUACGAUUGUAAAGACAUGUCAGACGAGGUCGGAUGCGUGAACGUCACGUUGUGCGAGGGUCCGAACAAGUUUAAGUGCCAUUCGGGCGAAUGUAUUACGCUCGAUAAAGUCUGCAACAUGGCGCGAGAUUGUAGGGAUUGGUCAGACGAACCCAUCAAGGAGUGCGGCACUAACGAGUGUUUGGACAAUAACGGCGGGUGUUCGCACGUCUGCAAUGAACUCAAAAUUGGGUAUGAGUGUCUCUGUCCUGACGGAUUCCAGCUGGUCGCGCAGCGCAGAUGCGAGGACAUCGACGAGUGCCAGGACCCCGACACAUGUUCGCAGUUGUGUGUCAACCUUGAAGGAGGGUACAAGUGCCAGUGCGAGGAGGGAUUUCAGCUUGACCCGCACACGAAAGCAUGUAAAGCGGUGGGGUCCAUUGCGUAUUUGUUUUUCACAAACAGACAUGAAGUGCGGAAGAUGACCCUUGAUCGCAGCGAAUAUACGUCACUGAUCCCUAAUCUUAGGAAUGUCGUGGCCCUUGACACGGAGGUAGCAUCAAAUAGAAUCUACUGGUCCGACCUCUCACAGAGAAUGAUCUGUUCAACACAGUUGGAUCGGGCGCACGGGGUGUCGUCGUACGAUACGGUAAUUAGCCGCGACAUCCAGGCGCCAGACGGACUCGCGGUCGACUGGAUCCAUAGCAACAUCUACUGGACAGACUCCGUGUUGGGAACCGUAUCCGUAGCUGACACAAAGGGAGUGAAGCGGAAAACUCUUUUUAGAGAGAACGGCAGCAAACCGAGAGCAAUCGUGGUCGAUCCGGUGCAUGGAUUCAUGUAUUGGACCGAUUGGGGAACGCCAGCCAAAAUCAAGAAAGGCGGUUUGAAUGGGGUCGACAUCUACUCGCUGGUGACUGAGAAUAUUCAGUGGCCAAACGGGAUCACCUUGGACUUGUUGUCGGGGAGGUUGUAUUGGGUGGACUCAAAGCUCCACUCGAUCAGCUCGAUCGACGUGAACGGCGGAAAUAGGAAAACUAUUCUCGAAGAUGAGAAAAGACUGGCCCACCCCUUCUCGCUCGCGGUGUUCGAGGACAAAGUAUUUUGGACAGACAUCAUCAACGAAGCGAUCUUUUCAGCCAACCGCCUGACAGGGUCGGAUGUCAAUCUCUUGGCCGAAAACCUUCUGAGCCCGGAAGAUAUGGUCUUGUUUCACAAUUUGACCCAACCCAGAGGUGUGAAUUGGUGCGAACGGACGACAUUGUCGAACGGAGGUUGCCAGUAUCUCUGUCUCCCUGCACCCCAGAUUAAUCCCCAUUCACCCAAGUUCACGUGUGCGUGCCCAGACGGAAUGCUUCUUGCGAGGGACAUGAGAUCCUGUCUCACCGAAGCGGAAGCGGCAGUGGCCACACAAGAGACUUCGACUGUCCGCCUUAAAGUGUCCUCGACGGCGGUCCGAACUCAGCAUACGACCACACGACCCGUGCCCGAUACCUCGCGGUUGCCCGGAGCAACACCGGGGUUGACGACAGUAGAAAUCGUAACCAUGAGCCACCAGGCACUUGGAGAUGUCGCAGGCAGAGGCAAUGAGAAGAAACCCAGCUCGGUCAGAGCCCUCAGCAUCGUGCUGCCUAUUGUGCUGCUUGUGUUUCUCUGUUUGGGUGUGUUCUUGUUGUGGAAGAACUGGCGCCUUAAGAAUAUCAACUCGAUUAACUUCGAUAAUCCGGUAUACCAGAAAACCACAGAGGAUGAAGUGCAUAUUUGUCACAACCAAGAUGGCUAUUCGUACCCGUCCAGGCAAAUGGUAUCACUUGAGGACGACGUGGCCUGAUAAUAGGCUGCCUUCUGCGGGGCUUGCCUUCUGGCCAUGCCCUUCUUCUCUCCCUUGCACCUGUACCUCUUGGUCUUUGAAUAAAGCCUGAGU AGGAAG LDLR1_L339DGGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAG  8 mRNA AGCCACCAUGGGUCCGUGGGGCUGGAAGCUUAGAUGGACAGUCGCGCUCCUCCUUGCAGCAGCAGGAACUGCGGUCGGAGAUCGAUGCGAGCGCAACGAGUUCCAAUGCCAAGAUGGGAAGUGUAUUUCGUACAAGUGGGUCUGCGAUGGAUCAGCGGAAUGUCAGGACGGAAGCGAUGAGAGCCAAGAAACAUGCCUCUCAGUGACAUGCAAGUCGGGAGACUUCUCGUGCGGAGGACGCGUAAACAGAUGUAUUCCACAGUUUUGGCGCUGCGAUGGUCAGGUGGACUGCGACAACGGUUCAGAUGAACAGGGAUGUCCUCCGAAAACGUGCUCACAAGACGAGUUUCGCUGCCAUGAUGGAAAGUGCAUUUCGCGGCAGUUCGUAUGUGAUUCGGAUCGGGACUGUCUGGACGGCUCGGACGAAGCGUCAUGCCCGGUACUUACUUGCGGGCCAGCCUCAUUCCAAUGCAACAGCUCAACGUGCAUUCCCCAGCUGUGGGCCUGUGACAAUGAUCCUGAUUGUGAGGACGGUAGCGACGAGUGGCCGCAGAGAUGUAGGGGUUUGUACGUAUUCCAAGGAGACUCAAGCCCCUGUUCCGCCUUUGAGUUUCACUGCCUGUCGGGUGAAUGCAUCCACUCCAGCUGGCGAUGUGAUGGUGGGCCCGACUGCAAAGAUAAGAGCGACGAGGAGAAUUGCGCGGUCGCGACGUGCAGACCCGAUGAGUUCCAGUGCUCCGAUGGAAACUGCAUCCACGGGAGCCGGCAGUGUGAUCGCGAGUACGAUUGUAAAGACAUGUCAGACGAGGUCGGAUGCGUGAACGUCACGUUGUGCGAGGGUCCGAACAAGUUUAAGUGCCAUUCGGGCGAAUGUAUUACGCUCGAUAAAGUCUGCAACAUGGCGCGAGAUUGUAGGGAUUGGUCAGACGAACCCAUCAAGGAGUGCGGCACUAACGAGUGUUUGGACAAUAACGGCGGGUGUUCGCACGUCUGCAAUGAUCUCAAAAUUGGGUAUGAGUGUGAUUGUCCUGACGGAUUCCAGCUGGUCGCGCAGCGCAGAUGCGAGGACAUCGACGAGUGCCAGGACCCCGACACAUGUUCGCAGUUGUGUGUCAACCUUGAAGGAGGGUACAAGUGCCAGUGCGAGGAGGGAUUUCAGCUUGACCCGCACACGAAAGCAUGUAAAGCGGUGGGGUCCAUUGCGUAUUUGUUUUUCACAAACAGACAUGAAGUGCGGAAGAUGACCCUUGAUCGCAGCGAAUAUACGUCACUGAUCCCUAAUCUUAGGAAUGUCGUGGCCCUUGACACGGAGGUAGCAUCAAAUAGAAUCUACUGGUCCGACCUCUCACAGAGAAUGAUCUGUUCAACACAGUUGGAUCGGGCGCACGGGGUGUCGUCGUACGAUACGGUAAUUAGCCGCGACAUCCAGGCGCCAGACGGACUCGCGGUCGACUGGAUCCAUAGCAACAUCUACUGGACAGACUCCGUGUUGGGAACCGUAUCCGUAGCUGACACAAAGGGAGUGAAGCGGAAAACUCUUUUUAGAGAGAACGGCAGCAAACCGAGAGCAAUCGUGGUCGAUCCGGUGCAUGGAUUCAUGUAUUGGACCGAUUGGGGAACGCCAGCCAAAAUCAAGAAAGGCGGUUUGAAUGGGGUCGACAUCUACUCGCUGGUGACUGAGAAUAUUCAGUGGCCAAACGGGAUCACCUUGGACUUGUUGUCGGGGAGGUUGUAUUGGGUGGACUCAAAGCUCCACUCGAUCAGCUCGAUCGACGUGAACGGCGGAAAUAGGAAAACUAUUCUCGAAGAUGAGAAAAGACUGGCCCACCCCUUCUCGCUCGCGGUGUUCGAGGACAAAGUAUUUUGGACAGACAUCAUCAACGAAGCGAUCUUUUCAGCCAACCGCCUGACAGGGUCGGAUGUCAAUCUCUUGGCCGAAAACCUUCUGAGCCCGGAAGAUAUGGUCUUGUUUCACAAUUUGACCCAACCCAGAGGUGUGAAUUGGUGCGAACGGACGACAUUGUCGAACGGAGGUUGCCAGUAUCUCUGUCUCCCUGCACCCCAGAUUAAUCCCCAUUCACCCAAGUUCACGUGUGCGUGCCCAGACGGAAUGCUUCUUGCGAGGGACAUGAGAUCCUGUCUCACCGAAGCGGAAGCGGCAGUGGCCACACAAGAGACUUCGACUGUCCGCCUUAAAGUGUCCUCGACGGCGGUCCGAACUCAGCAUACGACCACACGACCCGUGCCCGAUACCUCGCGGUUGCCCGGAGCAACACCGGGGUUGACGACAGUAGAAAUCGUAACCAUGAGCCACCAGGCACUUGGAGAUGUCGCAGGCAGAGGCAAUGAGAAGAAACCCAGCUCGGUCAGAGCCCUCAGCAUCGUGCUGCCUAUUGUGCUGCUUGUGUUUCUCUGUUUGGGUGUGUUCUUGUUGUGGAAGAACUGGCGCCUUAAGAAUAUCAACUCGAUUAACUUCGAUAAUCCGGUAUACCAGAAAACCACAGAGGAUGAAGUGCAUAUUUGUCACAACCAAGAUGGCUAUUCGUACCCGUCCAGGCAAAUGGUAUCACUUGAGGACGACGUGGCCUGAUAAUAGGCUGCCUUCUGCGGGGCUUGCCUUCUGGCCAUGCCCUUCUUCUCUCCCUUGCACCUGUACCUCUUGGUCUUUGAAUAAAGCCUGAGU AGGAAG LDLR1_N316AGGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAG  9 mRNA AGCCACCAUGGGUCCGUGGGGCUGGAAGCUUAGAUGGACAGUCGCGCUCCUCCUUGCAGCAGCAGGAACUGCGGUCGGAGAUCGAUGCGAGCGCAACGAGUUCCAAUGCCAAGAUGGGAAGUGUAUUUCGUACAAGUGGGUCUGCGAUGGAUCAGCGGAAUGUCAGGACGGAAGCGAUGAGAGCCAAGAAACAUGCCUCUCAGUGACAUGCAAGUCGGGAGACUUCUCGUGCGGAGGACGCGUAAACAGAUGUAUUCCACAGUUUUGGCGCUGCGAUGGUCAGGUGGACUGCGACAACGGUUCAGAUGAACAGGGAUGUCCUCCGAAAACGUGCUCACAAGACGAGUUUCGCUGCCAUGAUGGAAAGUGCAUUUCGCGGCAGUUCGUAUGUGAUUCGGAUCGGGACUGUCUGGACGGCUCGGACGAAGCGUCAUGCCCGGUACUUACUUGCGGGCCAGCCUCAUUCCAAUGCAACAGCUCAACGUGCAUUCCCCAGCUGUGGGCCUGUGACAAUGAUCCUGAUUGUGAGGACGGUAGCGACGAGUGGCCGCAGAGAUGUAGGGGUUUGUACGUAUUCCAAGGAGACUCAAGCCCCUGUUCCGCCUUUGAGUUUCACUGCCUGUCGGGUGAAUGCAUCCACUCCAGCUGGCGAUGUGAUGGUGGGCCCGACUGCAAAGAUAAGAGCGACGAGGAGAAUUGCGCGGUCGCGACGUGCAGACCCGAUGAGUUCCAGUGCUCCGAUGGAAACUGCAUCCACGGGAGCCGGCAGUGUGAUCGCGAGUACGAUUGUAAAGACAUGUCAGACGAGGUCGGAUGCGUGAACGUCACGUUGUGCGAGGGUCCGAACAAGUUUAAGUGCCAUUCGGGCGAAUGUAUUACGCUCGAUAAAGUCUGCAACAUGGCGCGAGAUUGUAGGGAUUGGUCAGACGAACCCAUCAAGGAGUGCGGCACUGCAGAGUGUUUGGACAAUAACGGCGGGUGUUCGCACGUCUGCAAUGAUCUCAAAAUUGGGUAUGAGUGUCUCUGUCCUGACGGAUUCCAGCUGGUCGCGCAGCGCAGAUGCGAGGACAUCGACGAGUGCCAGGACCCCGACACAUGUUCGCAGUUGUGUGUCAACCUUGAAGGAGGGUACAAGUGCCAGUGCGAGGAGGGAUUUCAGCUUGACCCGCACACGAAAGCAUGUAAAGCGGUGGGGUCCAUUGCGUAUUUGUUUUUCACAAACAGACAUGAAGUGCGGAAGAUGACCCUUGAUCGCAGCGAAUAUACGUCACUGAUCCCUAAUCUUAGGAAUGUCGUGGCCCUUGACACGGAGGUAGCAUCAAAUAGAAUCUACUGGUCCGACCUCUCACAGAGAAUGAUCUGUUCAACACAGUUGGAUCGGGCGCACGGGGUGUCGUCGUACGAUACGGUAAUUAGCCGCGACAUCCAGGCGCCAGACGGACUCGCGGUCGACUGGAUCCAUAGCAACAUCUACUGGACAGACUCCGUGUUGGGAACCGUAUCCGUAGCUGACACAAAGGGAGUGAAGCGGAAAACUCUUUUUAGAGAGAACGGCAGCAAACCGAGAGCAAUCGUGGUCGAUCCGGUGCAUGGAUUCAUGUAUUGGACCGAUUGGGGAACGCCAGCCAAAAUCAAGAAAGGCGGUUUGAAUGGGGUCGACAUCUACUCGCUGGUGACUGAGAAUAUUCAGUGGCCAAACGGGAUCACCUUGGACUUGUUGUCGGGGAGGUUGUAUUGGGUGGACUCAAAGCUCCACUCGAUCAGCUCGAUCGACGUGAACGGCGGAAAUAGGAAAACUAUUCUCGAAGAUGAGAAAAGACUGGCCCACCCCUUCUCGCUCGCGGUGUUCGAGGACAAAGUAUUUUGGACAGACAUCAUCAACGAAGCGAUCUUUUCAGCCAACCGCCUGACAGGGUCGGAUGUCAAUCUCUUGGCCGAAAACCUUCUGAGCCCGGAAGAUAUGGUCUUGUUUCACAAUUUGACCCAACCCAGAGGUGUGAAUUGGUGCGAACGGACGACAUUGUCGAACGGAGGUUGCCAGUAUCUCUGUCUCCCUGCACCCCAGAUUAAUCCCCAUUCACCCAAGUUCACGUGUGCGUGCCCAGACGGAAUGCUUCUUGCGAGGGACAUGAGAUCCUGUCUCACCGAAGCGGAAGCGGCAGUGGCCACACAAGAGACUUCGACUGUCCGCCUUAAAGUGUCCUCGACGGCGGUCCGAACUCAGCAUACGACCACACGACCCGUGCCCGAUACCUCGCGGUUGCCCGGAGCAACACCGGGGUUGACGACAGUAGAAAUCGUAACCAUGAGCCACCAGGCACUUGGAGAUGUCGCAGGCAGAGGCAAUGAGAAGAAACCCAGCUCGGUCAGAGCCCUCAGCAUCGUGCUGCCUAUUGUGCUGCUUGUGUUUCUCUGUUUGGGUGUGUUCUUGUUGUGGAAGAACUGGCGCCUUAAGAAUAUCAACUCGAUUAACUUCGAUAAUCCGGUAUACCAGAAAACCACAGAGGAUGAAGUGCAUAUUUGUCACAACCAAGAUGGCUAUUCGUACCCGUCCAGGCAAAUGGUAUCACUUGAGGACGACGUGGCCUGAUAAUAGGCUGCCUUCUGCGGGGCUUGCCUUCUGGCCAUGCCCUUCUUCUCUCCCUUGCACCUGUACCUCUUGGUCUUUGAAUAAAGCCUGAGU AGGAAG LDLR1_E317AGGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAG 10 mRNA AGCCACCAUGGGUCCGUGGGGCUGGAAGCUUAGAUGGACAGUCGCGCUCCUCCUUGCAGCAGCAGGAACUGCGGUCGGAGAUCGAUGCGAGCGCAACGAGUUCCAAUGCCAAGAUGGGAAGUGUAUUUCGUACAAGUGGGUCUGCGAUGGAUCAGCGGAAUGUCAGGACGGAAGCGAUGAGAGCCAAGAAACAUGCCUCUCAGUGACAUGCAAGUCGGGAGACUUCUCGUGCGGAGGACGCGUAAACAGAUGUAUUCCACAGUUUUGGCGCUGCGAUGGUCAGGUGGACUGCGACAACGGUUCAGAUGAACAGGGAUGUCCUCCGAAAACGUGCUCACAAGACGAGUUUCGCUGCCAUGAUGGAAAGUGCAUUUCGCGGCAGUUCGUAUGUGAUUCGGAUCGGGACUGUCUGGACGGCUCGGACGAAGCGUCAUGCCCGGUACUUACUUGCGGGCCAGCCUCAUUCCAAUGCAACAGCUCAACGUGCAUUCCCCAGCUGUGGGCCUGUGACAAUGAUCCUGAUUGUGAGGACGGUAGCGACGAGUGGCCGCAGAGAUGUAGGGGUUUGUACGUAUUCCAAGGAGACUCAAGCCCCUGUUCCGCCUUUGAGUUUCACUGCCUGUCGGGUGAAUGCAUCCACUCCAGCUGGCGAUGUGAUGGUGGGCCCGACUGCAAAGAUAAGAGCGACGAGGAGAAUUGCGCGGUCGCGACGUGCAGACCCGAUGAGUUCCAGUGCUCCGAUGGAAACUGCAUCCACGGGAGCCGGCAGUGUGAUCGCGAGUACGAUUGUAAAGACAUGUCAGACGAGGUCGGAUGCGUGAACGUCACGUUGUGCGAGGGUCCGAACAAGUUUAAGUGCCAUUCGGGCGAAUGUAUUACGCUCGAUAAAGUCUGCAACAUGGCGCGAGAUUGUAGGGAUUGGUCAGACGAACCCAUCAAGGAGUGCGGCACUAACGCAUGUUUGGACAAUAACGGCGGGUGUUCGCACGUCUGCAAUGAUCUCAAAAUUGGGUAUGAGUGUCUCUGUCCUGACGGAUUCCAGCUGGUCGCGCAGCGCAGAUGCGAGGACAUCGACGAGUGCCAGGACCCCGACACAUGUUCGCAGUUGUGUGUCAACCUUGAAGGAGGGUACAAGUGCCAGUGCGAGGAGGGAUUUCAGCUUGACCCGCACACGAAAGCAUGUAAAGCGGUGGGGUCCAUUGCGUAUUUGUUUUUCACAAACAGACAUGAAGUGCGGAAGAUGACCCUUGAUCGCAGCGAAUAUACGUCACUGAUCCCUAAUCUUAGGAAUGUCGUGGCCCUUGACACGGAGGUAGCAUCAAAUAGAAUCUACUGGUCCGACCUCUCACAGAGAAUGAUCUGUUCAACACAGUUGGAUCGGGCGCACGGGGUGUCGUCGUACGAUACGGUAAUUAGCCGCGACAUCCAGGCGCCAGACGGACUCGCGGUCGACUGGAUCCAUAGCAACAUCUACUGGACAGACUCCGUGUUGGGAACCGUAUCCGUAGCUGACACAAAGGGAGUGAAGCGGAAAACUCUUUUUAGAGAGAACGGCAGCAAACCGAGAGCAAUCGUGGUCGAUCCGGUGCAUGGAUUCAUGUAUUGGACCGAUUGGGGAACGCCAGCCAAAAUCAAGAAAGGCGGUUUGAAUGGGGUCGACAUCUACUCGCUGGUGACUGAGAAUAUUCAGUGGCCAAACGGGAUCACCUUGGACUUGUUGUCGGGGAGGUUGUAUUGGGUGGACUCAAAGCUCCACUCGAUCAGCUCGAUCGACGUGAACGGCGGAAAUAGGAAAACUAUUCUCGAAGAUGAGAAAAGACUGGCCCACCCCUUCUCGCUCGCGGUGUUCGAGGACAAAGUAUUUUGGACAGACAUCAUCAACGAAGCGAUCUUUUCAGCCAACCGCCUGACAGGGUCGGAUGUCAAUCUCUUGGCCGAAAACCUUCUGAGCCCGGAAGAUAUGGUCUUGUUUCACAAUUUGACCCAACCCAGAGGUGUGAAUUGGUGCGAACGGACGACAUUGUCGAACGGAGGUUGCCAGUAUCUCUGUCUCCCUGCACCCCAGAUUAAUCCCCAUUCACCCAAGUUCACGUGUGCGUGCCCAGACGGAAUGCUUCUUGCGAGGGACAUGAGAUCCUGUCUCACCGAAGCGGAAGCGGCAGUGGCCACACAAGAGACUUCGACUGUCCGCCUUAAAGUGUCCUCGACGGCGGUCCGAACUCAGCAUACGACCACACGACCCGUGCCCGAUACCUCGCGGUUGCCCGGAGCAACACCGGGGUUGACGACAGUAGAAAUCGUAACCAUGAGCCACCAGGCACUUGGAGAUGUCGCAGGCAGAGGCAAUGAGAAGAAACCCAGCUCGGUCAGAGCCCUCAGCAUCGUGCUGCCUAUUGUGCUGCUUGUGUUUCUCUGUUUGGGUGUGUUCUUGUUGUGGAAGAACUGGCGCCUUAAGAAUAUCAACUCGAUUAACUUCGAUAAUCCGGUAUACCAGAAAACCACAGAGGAUGAAGUGCAUAUUUGUCACAACCAAGAUGGCUAUUCGUACCCGUCCAGGCAAAUGGUAUCACUUGAGGACGACGUGGCCUGAUAAUAGGCUGCCUUCUGCGGGGCUUGCCUUCUGGCCAUGCCCUUCUUCUCUCCCUUGCACCUGUACCUCUUGGUCUUUGAAUAAAGCCUGAGU AGGAAG LDLR1_Y336AGGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAG 11 mRNA AGCCACCAUGGGUCCGUGGGGCUGGAAGCUUAGAUGGACAGUCGCGCUCCUCCUUGCAGCAGCAGGAACUGCGGUCGGAGAUCGAUGCGAGCGCAACGAGUUCCAAUGCCAAGAUGGGAAGUGUAUUUCGUACAAGUGGGUCUGCGAUGGAUCAGCGGAAUGUCAGGACGGAAGCGAUGAGAGCCAAGAAACAUGCCUCUCAGUGACAUGCAAGUCGGGAGACUUCUCGUGCGGAGGACGCGUAAACAGAUGUAUUCCACAGUUUUGGCGCUGCGAUGGUCAGGUGGACUGCGACAACGGUUCAGAUGAACAGGGAUGUCCUCCGAAAACGUGCUCACAAGACGAGUUUCGCUGCCAUGAUGGAAAGUGCAUUUCGCGGCAGUUCGUAUGUGAUUCGGAUCGGGACUGUCUGGACGGCUCGGACGAAGCGUCAUGCCCGGUACUUACUUGCGGGCCAGCCUCAUUCCAAUGCAACAGCUCAACGUGCAUUCCCCAGCUGUGGGCCUGUGACAAUGAUCCUGAUUGUGAGGACGGUAGCGACGAGUGGCCGCAGAGAUGUAGGGGUUUGUACGUAUUCCAAGGAGACUCAAGCCCCUGUUCCGCCUUUGAGUUUCACUGCCUGUCGGGUGAAUGCAUCCACUCCAGCUGGCGAUGUGAUGGUGGGCCCGACUGCAAAGAUAAGAGCGACGAGGAGAAUUGCGCGGUCGCGACGUGCAGACCCGAUGAGUUCCAGUGCUCCGAUGGAAACUGCAUCCACGGGAGCCGGCAGUGUGAUCGCGAGUACGAUUGUAAAGACAUGUCAGACGAGGUCGGAUGCGUGAACGUCACGUUGUGCGAGGGUCCGAACAAGUUUAAGUGCCAUUCGGGCGAAUGUAUUACGCUCGAUAAAGUCUGCAACAUGGCGCGAGAUUGUAGGGAUUGGUCAGACGAACCCAUCAAGGAGUGCGGCACUAACGAGUGUUUGGACAAUAACGGCGGGUGUUCGCACGUCUGCAAUGAUCUCAAAAUUGGGGCAGAGUGUCUCUGUCCUGACGGAUUCCAGCUGGUCGCGCAGCGCAGAUGCGAGGACAUCGACGAGUGCCAGGACCCCGACACAUGUUCGCAGUUGUGUGUCAACCUUGAAGGAGGGUACAAGUGCCAGUGCGAGGAGGGAUUUCAGCUUGACCCGCACACGAAAGCAUGUAAAGCGGUGGGGUCCAUUGCGUAUUUGUUUUUCACAAACAGACAUGAAGUGCGGAAGAUGACCCUUGAUCGCAGCGAAUAUACGUCACUGAUCCCUAAUCUUAGGAAUGUCGUGGCCCUUGACACGGAGGUAGCAUCAAAUAGAAUCUACUGGUCCGACCUCUCACAGAGAAUGAUCUGUUCAACACAGUUGGAUCGGGCGCACGGGGUGUCGUCGUACGAUACGGUAAUUAGCCGCGACAUCCAGGCGCCAGACGGACUCGCGGUCGACUGGAUCCAUAGCAACAUCUACUGGACAGACUCCGUGUUGGGAACCGUAUCCGUAGCUGACACAAAGGGAGUGAAGCGGAAAACUCUUUUUAGAGAGAACGGCAGCAAACCGAGAGCAAUCGUGGUCGAUCCGGUGCAUGGAUUCAUGUAUUGGACCGAUUGGGGAACGCCAGCCAAAAUCAAGAAAGGCGGUUUGAAUGGGGUCGACAUCUACUCGCUGGUGACUGAGAAUAUUCAGUGGCCAAACGGGAUCACCUUGGACUUGUUGUCGGGGAGGUUGUAUUGGGUGGACUCAAAGCUCCACUCGAUCAGCUCGAUCGACGUGAACGGCGGAAAUAGGAAAACUAUUCUCGAAGAUGAGAAAAGACUGGCCCACCCCUUCUCGCUCGCGGUGUUCGAGGACAAAGUAUUUUGGACAGACAUCAUCAACGAAGCGAUCUUUUCAGCCAACCGCCUGACAGGGUCGGAUGUCAAUCUCUUGGCCGAAAACCUUCUGAGCCCGGAAGAUAUGGUCUUGUUUCACAAUUUGACCCAACCCAGAGGUGUGAAUUGGUGCGAACGGACGACAUUGUCGAACGGAGGUUGCCAGUAUCUCUGUCUCCCUGCACCCCAGAUUAAUCCCCAUUCACCCAAGUUCACGUGUGCGUGCCCAGACGGAAUGCUUCUUGCGAGGGACAUGAGAUCCUGUCUCACCGAAGCGGAAGCGGCAGUGGCCACACAAGAGACUUCGACUGUCCGCCUUAAAGUGUCCUCGACGGCGGUCCGAACUCAGCAUACGACCACACGACCCGUGCCCGAUACCUCGCGGUUGCCCGGAGCAACACCGGGGUUGACGACAGUAGAAAUCGUAACCAUGAGCCACCAGGCACUUGGAGAUGUCGCAGGCAGAGGCAAUGAGAAGAAACCCAGCUCGGUCAGAGCCCUCAGCAUCGUGCUGCCUAUUGUGCUGCUUGUGUUUCUCUGUUUGGGUGUGUUCUUGUUGUGGAAGAACUGGCGCCUUAAGAAUAUCAACUCGAUUAACUUCGAUAAUCCGGUAUACCAGAAAACCACAGAGGAUGAAGUGCAUAUUUGUCACAACCAAGAUGGCUAUUCGUACCCGUCCAGGCAAAUGGUAUCACUUGAGGACGACGUGGCCUGAUAAUAGGCUGCCUUCUGCGGGGCUUGCCUUCUGGCCAUGCCCUUCUUCUCUCCCUUGCACCUGUACCUCUUGGUCUUUGAAUAAAGCCUGAGU AGGAAG LDLR1_4AGGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAG 12 mRNA AGCCACCAUGGGUCCGUGGGGCUGGAAGCUUAGAUGGACAGUCGCGCUCCUCCUUGCAGCAGCAGGAACUGCGGUCGGAGAUCGAUGCGAGCGCAACGAGUUCCAAUGCCAAGAUGGGAAGUGUAUUUCGUACAAGUGGGUCUGCGAUGGAUCAGCGGAAUGUCAGGACGGAAGCGAUGAGAGCCAAGAAACAUGCCUCUCAGUGACAUGCAAGUCGGGAGACUUCUCGUGCGGAGGACGCGUAAACAGAUGUAUUCCACAGUUUUGGCGCUGCGAUGGUCAGGUGGACUGCGACAACGGUUCAGAUGAACAGGGAUGUCCUCCGAAAACGUGCUCACAAGACGAGUUUCGCUGCCAUGAUGGAAAGUGCAUUUCGCGGCAGUUCGUAUGUGAUUCGGAUCGGGACUGUCUGGACGGCUCGGACGAAGCGUCAUGCCCGGUACUUACUUGCGGGCCAGCCUCAUUCCAAUGCAACAGCUCAACGUGCAUUCCCCAGCUGUGGGCCUGUGACAAUGAUCCUGAUUGUGAGGACGGUAGCGACGAGUGGCCGCAGAGAUGUAGGGGUUUGUACGUAUUCCAAGGAGACUCAAGCCCCUGUUCCGCCUUUGAGUUUCACUGCCUGUCGGGUGAAUGCAUCCACUCCAGCUGGCGAUGUGAUGGUGGGCCCGACUGCAAAGAUAAGAGCGACGAGGAGAAUUGCGCGGUCGCGACGUGCAGACCCGAUGAGUUCCAGUGCUCCGAUGGAAACUGCAUCCACGGGAGCCGGCAGUGUGAUCGCGAGUACGAUUGUAAAGACAUGUCAGACGAGGUCGGAUGCGUGAACGUCACGUUGUGCGAGGGUCCGAACAAGUUUAAGUGCCAUUCGGGCGAAUGUAUUACGCUCGAUAAAGUCUGCAACAUGGCGCGAGAUUGUAGGGAUUGGUCAGACGAACCCAUCAAGGAGUGCGGCACUGCAGCAUGUUUGGACAAUAACGGCGGGUGUUCGCACGUCUGCAAUGCACUCAAAAUUGGGGCAGAGUGUCUCUGUCCUGACGGAUUCCAGCUGGUCGCGCAGCGCAGAUGCGAGGACAUCGACGAGUGCCAGGACCCCGACACAUGUUCGCAGUUGUGUGUCAACCUUGAAGGAGGGUACAAGUGCCAGUGCGAGGAGGGAUUUCAGCUUGACCCGCACACGAAAGCAUGUAAAGCGGUGGGGUCCAUUGCGUAUUUGUUUUUCACAAACAGACAUGAAGUGCGGAAGAUGACCCUUGAUCGCAGCGAAUAUACGUCACUGAUCCCUAAUCUUAGGAAUGUCGUGGCCCUUGACACGGAGGUAGCAUCAAAUAGAAUCUACUGGUCCGACCUCUCACAGAGAAUGAUCUGUUCAACACAGUUGGAUCGGGCGCACGGGGUGUCGUCGUACGAUACGGUAAUUAGCCGCGACAUCCAGGCGCCAGACGGACUCGCGGUCGACUGGAUCCAUAGCAACAUCUACUGGACAGACUCCGUGUUGGGAACCGUAUCCGUAGCUGACACAAAGGGAGUGAAGCGGAAAACUCUUUUUAGAGAGAACGGCAGCAAACCGAGAGCAAUCGUGGUCGAUCCGGUGCAUGGAUUCAUGUAUUGGACCGAUUGGGGAACGCCAGCCAAAAUCAAGAAAGGCGGUUUGAAUGGGGUCGACAUCUACUCGCUGGUGACUGAGAAUAUUCAGUGGCCAAACGGGAUCACCUUGGACUUGUUGUCGGGGAGGUUGUAUUGGGUGGACUCAAAGCUCCACUCGAUCAGCUCGAUCGACGUGAACGGCGGAAAUAGGAAAACUAUUCUCGAAGAUGAGAAAAGACUGGCCCACCCCUUCUCGCUCGCGGUGUUCGAGGACAAAGUAUUUUGGACAGACAUCAUCAACGAAGCGAUCUUUUCAGCCAACCGCCUGACAGGGUCGGAUGUCAAUCUCUUGGCCGAAAACCUUCUGAGCCCGGAAGAUAUGGUCUUGUUUCACAAUUUGACCCAACCCAGAGGUGUGAAUUGGUGCGAACGGACGACAUUGUCGAACGGAGGUUGCCAGUAUCUCUGUCUCCCUGCACCCCAGAUUAAUCCCCAUUCACCCAAGUUCACGUGUGCGUGCCCAGACGGAAUGCUUCUUGCGAGGGACAUGAGAUCCUGUCUCACCGAAGCGGAAGCGGCAGUGGCCACACAAGAGACUUCGACUGUCCGCCUUAAAGUGUCCUCGACGGCGGUCCGAACUCAGCAUACGACCACACGACCCGUGCCCGAUACCUCGCGGUUGCCCGGAGCAACACCGGGGUUGACGACAGUAGAAAUCGUAACCAUGAGCCACCAGGCACUUGGAGAUGUCGCAGGCAGAGGCAAUGAGAAGAAACCCAGCUCGGUCAGAGCCCUCAGCAUCGUGCUGCCUAUUGUGCUGCUUGUGUUUCUCUGUUUGGGUGUGUUCUUGUUGUGGAAGAACUGGCGCCUUAAGAAUAUCAACUCGAUUAACUUCGAUAAUCCGGUAUACCAGAAAACCACAGAGGAUGAAGUGCAUAUUUGUCACAACCAAGAUGGCUAUUCGUACCCGUCCAGGCAAAUGGUAUCACUUGAGGACGACGUGGCCUGAUAAUAGGCUGCCUUCUGCGGGGCUUGCCUUCUGGCCAUGCCCUUCUUCUCUCCCUUGCACCUGUACCUCUUGGUCUUUGAAUAAAGCCUGAGU AGGAAG CommonGGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAG 31 LDLR1 5′UTR CCACC CommonTGATAATAGGCTGCCTTCTGCGGGGCTTGCCTTCTGGCCATGCC 32 LDLR1 3′UTRCTTCTTCTCTCCCTTGCACCTGTACCTCTTGGTCTTTGAATAAAG (mouse origin)CCTGAGTAGGAAG

Example 12. LDLR1 Protein Sequences

Sequences of the one or more mutan LDL-R1 proteins are given in Table 5.

TABLE 5 Protein sequences SEQ ID Description Sequence NO LDLR1_D331EMGPWGWKLRWTVALLLAAAGTAVGDRCERNEFQCQDGKCISY 33 ProteinKWVCDGSAECQDGSDESQETCLSVTCKSGDFSCGGRVNRCIPQFWRCDGQVDCDNGSDEQGCPPKTCSQDEFRCHDGKCISRQFVCDSDRDCLDGSDEASCPVLTCGPASFQCNSSTCIPQLWACDNDPDCEDGSDEWPQRCRGLYVFQGDSSPCSAFEFHCLSGECIHSSWRCDGGPDCKDKSDEENCAVATCRPDEFQCSDGNCIHGSRQCDREYDCKDMSDEVGCVNVTLCEGPNKFKCHSGECITLDKVCNMARDCRDWSDEPIKECGTNECLDNNGGCSHVCNELKIGYECLCPDGFQLVAQRRCEDIDECQDPDTCSQLCVNLEGGYKCQCEEGFQLDPHTKACKAVGSIAYLFFTNRHEVRKMTLDRSEYTSLIPNLRNVVALDTEVASNRIYWSDLSQRMICSTQLDRAHGVSSYDTVISRDIQAPDGLAVDWIHSNIYWTDSVLGTVSVADTKGVKRKTLFRENGSKPRAIVVDPVHGFMYWTDWGTPAKIKKGGLNGVDIYSLVTENIQWPNGITLDLLSGRLYWVDSKLHSISSIDVNGGNRKTILEDEKRLAHPFSLAVFEDKVFWTDIINEAIFSANRLTGSDVNLLAENLLSPEDMVLFHNLTQPRGVNWCERTTLSNGGCQYLCLPAPQINPHSPKFTCACPDGMLLARDMRSCLTEAEAAVATQETSTVRLKVSSTAVRTQHTTTRPVPDTSRLPGATPGLTTVEIVTMSHQALGDVAGRGNEKKPSSVRALSIVLPIVLLVFLCLGVFLLWKNWRLKNINSINFDNPVYQKTTEDEVHICHNQ DGYSYPSRQMVSLEDDVALDLR1_L339D MGPWGWKLRWTVALLLAAAGTAVGDRCERNEFQCQDGKCISY 34 ProteinKWVCDGSAECQDGSDESQETCLSVTCKSGDFSCGGRVNRCIPQFWRCDGQVDCDNGSDEQGCPPKTCSQDEFRCHDGKCISRQFVCDSDRDCLDGSDEASCPVLTCGPASFQCNSSTCIPQLWACDNDPDCEDGSDEWPQRCRGLYVFQGDSSPCSAFEFHCLSGECIHSSWRCDGGPDCKDKSDEENCAVATCRPDEFQCSDGNCIHGSRQCDREYDCKDMSDEVGCVNVTLCEGPNKFKCHSGECITLDKVCNMARDCRDWSDEPIKECGTNECLDNNGGCSHVCNDLKIGYECDCPDGFQLVAQRRCEDIDECQDPDTCSQLCVNLEGGYKCQCEEGFQLDPHTKACKAVGSIAYLFFTNRHEVRKMTLDRSEYTSLIPNLRNVVALDIEVASNRIYWSDLSQRMICSTQLDRAHGVSSYDTVISRDIQAPDGLAVDWIHSNIYWTDSVLGTVSVADTKGVKRKTLFRENGSKPRAIVVDPVHGFMYWTDWGTPAKIKKGGLNGVDIYSLVTENIQWPNGITLDLLSGRLYWVDSKLHSISSIDVNGGNRKTILEDEKRLAHPFSLAVFEDKVFWTDIINEAIFSANRLTGSDVNLLAENLLSPEDMVLFHNLTQPRGVNWCERTTLSNGGCQYLCLPAPQINPHSPKFTCACPDGMLLARDMRSCLIEAEAAVATQETSTVRLKVSSTAVRTQHTTTRPVPDTSRLPGATPGLTTVEIVTMSHQALGDVAGRGNEKKPSSVRALSIVLPIVLLVFLCLGVFLLWKNWRLKNINSINFDNPVYQKTIEDEVHICH NQDGYSYPSRQMVSLEDDVALDLR1_N316A MGPWGWKLRWTVALLLAAAGTAVGDRCERNEFQCQDGKCISY 35 ProteinKWVCDGSAECQDGSDESQETCLSVTCKSGDFSCGGRVNRCIPQFWRCDGQVDCDNGSDEQGCPPKTCSQDEFRCHDGKCISRQFVCDSDRDCLDGSDEASCPVLTCGPASFQCNSSTCIPQLWACDNDPDCEDGSDEWPQRCRGLYVFQGDSSPCSAFEFHCLSGECIHSSWRCDGGPDCKDKSDEENCAVATCRPDEFQCSDGNCIHGSRQCDREYDCKDMSDEVGCVNVTLCEGPNKFKCHSGECITLDKVCNMARDCRDWSDEPIKECGTAECLDNNGGCSHVCNDLKIGYECLCPDGFQLVAQRRCEDIDECQDPDTCSQLCVNLEGGYKCQCEEGFQLDPHTKACKAVGSIAYLFFTNRHEVRKMTLDRSEYTSLIPNLRNVVALDIEVASNRIYWSDLSQRMICSTQLDRAHGVSSYDTVISRDIQAPDGLAVDWIHSNIYWTDSVLGTVSVADTKGVKRKTLFRENGSKPRAIVVDPVHGFMYWTDWGTPAKIKKGGLNGVDIYSLVTENIQWPNGITLDLLSGRLYWVDSKLHSISSIDVNGGNRKTILEDEKRLAHPFSLAVFEDKVFWTDIINEAIFSANRLTGSDVNLLAENLLSPEDMVLFHNLTQPRGVNWCERTTLSNGGCQYLCLPAPQINPHSPKFTCACPDGMLLARDMRSCLIEAEAAVATQETSTVRLKVSSTAVRTQHTTTRPVPDTSRLPGATPGLTTVEIVTMSHQALGDVAGRGNEKKPSSVRALSIVLPIVLLVFLCLGVFLLWKNWRLKNINSINFDNPVYQKTIEDEVHICH NQDGYSYPSRQMVSLEDDVALDLR1_E317A MGPWGWKLRWTVALLLAAAGTAVGDRCERNEFQCQDGKCISY 36 ProteinKWVCDGSAECQDGSDESQETCLSVTCKSGDFSCGGRVNRCIPQFWRCDGQVDCDNGSDEQGCPPKTCSQDEFRCHDGKCISRQFVCDSDRDCLDGSDEASCPVLTCGPASFQCNSSTCIPQLWACDNDPDCEDGSDEWPQRCRGLYVFQGDSSPCSAFEFHCLSGECIHSSWRCDGGPDCKDKSDEENCAVATCRPDEFQCSDGNCIHGSRQCDREYDCKDMSDEVGCVNVTLCEGPNKFKCHSGECITLDKVCNMARDCRDWSDEPIKECGTNACLDNNGGCSHVCNDLKIGYECLCPDGFQLVAQRRCEDIDECQDPDTCSQLCVNLEGGYKCQCEEGFQLDPHTKACKAVGSIAYLFFTNRHEVRKMTLDRSEYTSLIPNLRNVVALDIEVASNRIYWSDLSQRMICSTQLDRAHGVSSYDTVISRDIQAPDGLAVDWIHSNIYWTDSVLGTVSVADTKGVKRKTLFRENGSKPRAIVVDPVHGFMYWTDWGTPAKIKKGGLNGVDIYSLVTENIQWPNGITLDLLSGRLYWVDSKLHSISSIDVNGGNRKTILEDEKRLAHPFSLAVFEDKVFWTDIINEAIFSANRLTGSDVNLLAENLLSPEDMVLFHNLTQPRGVNWCERTTLSNGGCQYLCLPAPQINPHSPKFTCACPDGMLLARDMRSCLIEAEAAVATQETSTVRLKVSSTAVRTQHTTTRPVPDTSRLPGATPGLTTVEIVTMSHQALGDVAGRGNEKKPSSVRALSIVLPIVLLVFLCLGVFLLWKNWRLKNINSINFDNPVYQKTIEDEVHICH NQDGYSYPSRQMVSLEDDVALDLR1_Y336A MGPWGWKLRWTVALLLAAAGTAVGDRCERNEFQCQDGKCISY 37 ProteinKWVCDGSAECQDGSDESQETCLSVTCKSGDFSCGGRVNRCIPQFWRCDGQVDCDNGSDEQGCPPKTCSQDEFRCHDGKCISRQFVCDSDRDCLDGSDEASCPVLTCGPASFQCNSSTCIPQLWACDNDPDCEDGSDEWPQRCRGLYVFQGDSSPCSAFEFHCLSGECIHSSWRCDGGPDCKDKSDEENCAVATCRPDEFQCSDGNCIHGSRQCDREYDCKDMSDEVGCVNVTLCEGPNKFKCHSGECITLDKVCNMARDCRDWSDEPIKECGTNECLDNNGGCSHVCNDLKIGAECLCPDGFQLVAQRRCEDIDECQDPDTCSQLCVNLEGGYKCQCEEGFQLDPHTKACKAVGSIAYLFFTNRHEVRKMTLDRSEYTSLIPNLRNVVALDIEVASNRIYWSDLSQRMICSTQLDRAHGVSSYDTVISRDIQAPDGLAVDWIHSNIYWTDSVLGTVSVADTKGVKRKTLFRENGSKPRAIVVDPVHGFMYWTDWGTPAKIKKGGLNGVDIYSLVTENIQWPNGITLDLLSGRLYWVDSKLHSISSIDVNGGNRKTILEDEKRLAHPFSLAVFEDKVFWTDIINEAIFSANRLTGSDVNLLAENLLSPEDMVLFHNLTQPRGVNWCERTTLSNGGCQYLCLPAPQINPHSPKFTCACPDGMLLARDMRSCLIEAEAAVATQETSTVRLKVSSTAVRTQHTTTRPVPDTSRLPGATPGLTTVEIVTMSHQALGDVAGRGNEKKPSSVRALSIVLPIVLLVFLCLGVFLLWKNWRLKNINSINFDNPVYQKTIEDEVHICH NQDGYSYPSRQMVSLEDDVALDLR1_4A MGPWGWKLRWTVALLLAAAGTAVGDRCERNEFQCQDGKCISY 38 ProteinKWVCDGSAECQDGSDESQETCLSVTCKSGDFSCGGRVNRCIPQFWRCDGQVDCDNGSDEQGCPPKTCSQDEFRCHDGKCISRQFVCDSDRDCLDGSDEASCPVLTCGPASFQCNSSTCIPQLWACDNDPDCEDGSDEWPQRCRGLYVFQGDSSPCSAFEFHCLSGECIHSSWRCDGGPDCKDKSDEENCAVATCRPDEFQCSDGNCIHGSRQCDREYDCKDMSDEVGCVNVTLCEGPNKFKCHSGECITLDKVCNMARDCRDWSDEPIKECGTAACLDNNGGCSHVCNALKIGAECLCPDGFQLVAQRRCEDIDECQDPDTCSQLCVNLEGGYKCQCEEGFQLDPHTKACKAVGSIAYLFFTNRHEVRKMTLDRSEYTSLIPNLRNVVALDIEVASNRIYWSDLSQRMICSTQLDRAHGVSSYDTVISRDIQAPDGLAVDWIHSNIYWTDSVLGTVSVADTKGVKRKTLFRENGSKPRAIVVDPVHGFMYWTDWGTPAKIKKGGLNGVDIYSLVTENIQWPNGITLDLLSGRLYWVDSKLHSISSIDVNGGNRKTILEDEKRLAHPFSLAVFEDKVFWTDIINEAIFSANRLTGSDVNLLAENLLSPEDMVLFHNLTQPRGVNWCERTTLSNGGCQYLCLPAPQINPHSPKFTCACPDGMLLARDMRSCLIEAEAAVATQETSTVRLKVSSTAVRTQHTTTRPVPDTSRLPGATPGLTTVEIVTMSHQALGDVAGRGNEKKPSSVRALSIVLPIVLLVFLCLGVFLLWKNWRLKNINSINFDNPVYQKTIEDEVHICH NQDGYSYPSRQMVSLEDDVA

Example 13. Cyp7a1 Sequence

Sequences encoding CYP7A1 protein open reading frame, and the 5′UTR and3′UTR are given in Table 6. Also shown is the sequence of the encodedprotein.

TABLE 6 CYP7a1 sequences SEQ Description Sequence ID NO CYP7a1ATGATGACCACATCTTTGATTTGGGGGATTGCTATAGCAGCA 39 Coding RegionTGCTGTTGTCTATGGCTTATTCTTGGAATTAGGAGAAGGCAAACGGGTGAACCACCTCTTGAGAATGGATTAATTCCATACCTGGGCTGTGCTCTGCAATTTGGTGCCAATCCTCTTGAGTTCCTCAGAGCAAATCAAAGGAAACATGGTCATGTTTTTACCTGCAAACTAATGGGAAAATATGTCCATTTCATCACAAATCCCTTGTCATACCATAAGGTGTTGTGCCACGGAAAATATTTTGATTGGAAAAAATTTCACTTTGCTACTTCTGCGAAGGCATTTGGGCACAGAAGCATTGACCCGATGGATGGAAATACCACTGAAAACATAAACGACACTTTCATCAAAACCCTGCAGGGCCATGCCTTGAATTCCCTCACGGAAAGCATGATGGAAAACCTCCAACGTATCATGAGACCTCCAGTCTCCTCTAACTCAAAGACCGCTGCCTGGGTGACAGAAGGGATGTATTCTTTCTGCTACCGAGTGATGTTTGAAGCTGGGTATTTAACTATCTTTGGCAGAGATCTTACAAGGCGGGACACACAGAAAGCACATATTCTAAACAATCTTGACAACTTCAAGCAATTCGACAAAGTCTTTCCAGCCCTGGTAGCAGGCCTCCCCATTCACATGTTCAGGACTGCGCACAATGCCCGGGAGAAACTGGCAGAGAGCTTGAGGCACGAGAACCTCCAAAAGAGGGAAAGCATCTCAGAACTGATCAGCCTGCGCATGTTTCTCAATGACACTTTGTCCACCTTTGATGATCTGGAGAAGGCCAAGACACACCTCGTGGTCCTCTGGGCATCGCAAGCAAACACCATTCCAGCGACTTTCTGGAGTTTATTTCAAATGATTAGGAACCCAGAAGCAATGAAAGCAGCTACTGAAGAAGTGAAAAGAACATTAGAGAATGCTGGTCAAAAAGTCAGCTTGGAAGGCAATCCTATTTGTTTGAGTCAAGCAGAACTGAATGACCTGCCAGTATTAGATAGTATAATCAAGGAATCGCTGAGGCTTTCCAGTGCCTCCCTCAACATCCGGACAGCTAAGGAGGATTTCACTTTGCACCTTGAGGACGGTTCCTACAACATCCGAAAAGATGACATCATAGCTCTTTACCCACAGTTAATGCACTTAGATCCAGAAATCTACCCAGACCCTTTGACTTTTAAATATGATAGGTATCTTGATGAAAACGGGAAGACAAAGACTACCTTCTATTGTAATGGACTCAAGTTAAAGTATTACTACATGCCCTTTGGATCGGGAGCTACAATATGTCCTGGAAGATTGTTCGCTATCCACGAAATCAAGCAATTTTTGATTCTGATGCTTTCTTATTTTGAATTGGAGCTTATAGAGGGCCAAGCTAAATGTCCACCTTTGGACCAGTCCCGGGCAGGCTTGGGCATTTTGCCGCCATTGAATGATATTGAATTTAAATATAAA TTCAAGCATTTG CYP7a1GGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAG 40 5 ′UTR AGCCACC CYP7a1TGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCT 41 3 ′UTRTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC CYP7A1MMTTSLIWGIAIAACCCLWLILGIRRRQTGEPPLENGLIPYLGCA 23 ProteinLQFGANPLEFLRANQRKHGHVFTCKLMGKYVHFITNPLSYHKVLCHGKYFDWKKFHFATSAKAFGHRSIDPMDGNTTENINDTFIKTLQGHALNSLTESMMENLQRIMRPPVSSNSKTAAWVTEGMYSFCYRVMFEAGYLTIFGRDLTRRDTQKAHILNNLDNFKQFDKVFPALVAGLPIHMFRTAHNAREKLAESLRHENLQKRESISELISLRMFLNDTLSTFDDLEKAKTHLVVLWASQANTIPATFWSLFQMIRNPEAMKAATEEVKRTLENAGQKVSLEGNPICLSQAELNDLPVLDSIIKESLRLSSASLNIRTAKEDFTLHLEDGSYNIRKDDIIALYPQLMHLDPEIYPDPLTFKYDRYLDENGKTKTTFYCNGLKLKYYYMPFGSGATICPGRLFAIHEIKQFLILMLSYFELELIEGQAKCPPLD QSRAGLGILPPLNDIEFKYKFKHL

Example 14. NASH HCC Animal Model

Compounds including polynucleotides, primary constructs and mmRNA of theinvention may be tested in animal models for non-alcololicsteatohepatitis (NASH). One model involves the use of STAM™ mice (StelicInstitute and Co, Tokyo Japan).

Materials for Examples 15-20

Sequences encoding LDLR protein open reading frame, mCherry protein openreading frame, and luciferase protein open reading frame and the 5′UTRand 3′UTR are given in Table 7. The start site of the RNA is underlined“AUG” and the 5′ UTR as well as the 3′UTR are bolded.

TABLE 7 LDLR, mCherry and Luciferase Sequences SEQ Name Sequence ID NOLDLR GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCAC 42 mRNA CAUGGGUCCGUGGGGCUGGAAGCUUAGAUGGACAGUCGCGCUCCUCCU sequenceUGCAGCAGCAGGAACUGCGGUCGGAGAUCGAUGCGAGCGCAACGAGUUCCAAUGCCAAGAUGGGAAGUGUAUUUCGUACAAGUGGGUCUGCGAUGGAUCAGCGGAAUGUCAGGACGGAAGCGAUGAGAGCCAAGAAACAUGCCUCUCAGUGACAUGCAAGUCGGGAGACUUCUCGUGCGGAGGACGCGUAAACAGAUGUAUUCCACAGUUUUGGCGCUGCGAUGGUCAGGUGGACUGCGACAACGGUUCAGAUGAACAGGGAUGUCCUCCGAAAACGUGCUCACAAGACGAGUUUCGCUGCCAUGAUGGAAAGUGCAUUUCGCGGCAGUUCGUAUGUGAUUCGGAUCGGGACUGUCUGGACGGCUCGGACGAAGCGUCAUGCCCGGUACUUACUUGCGGGCCAGCCUCAUUCCAAUGCAACAGCUCAACGUGCAUUCCCCAGCUGUGGGCCUGUGACAAUGAUCCUGAUUGUGAGGACGGUAGCGACGAGUGGCCGCAGAGAUGUAGGGGUUUGUACGUAUUCCAAGGAGACUCAAGCCCCUGUUCCGCCUUUGAGUUUCACUGCCUGUCGGGUGAAUGCAUCCACUCCAGCUGGCGAUGUGAUGGUGGGCCCGACUGCAAAGAUAAGAGCGACGAGGAGAAUUGCGCGGUCGCGACGUGCAGACCCGAUGAGUUCCAGUGCUCCGAUGGAAACUGCAUCCACGGGAGCCGGCAGUGUGAUCGCGAGUACGAUUGUAAAGACAUGUCAGACGAGGUCGGAUGCGUGAACGUCACGUUGUGCGAGGGUCCGAACAAGUUUAAGUGCCAUUCGGGCGAAUGUAUUACGCUCGAUAAAGUCUGCAACAUGGCGCGAGAUUGUAGGGAUUGGUCAGACGAACCCAUCAAGGAGUGCGGCACUAACGAGUGUUUGGACAAUAACGGCGGGUGUUCGCACGUCUGCAAUGAUCUCAAAAUUGGGUAUGAGUGUCUCUGUCCUGACGGAUUCCAGCUGGUCGCGCAGCGCAGAUGCGAGGACAUCGACGAGUGCCAGGACCCCGACACAUGUUCGCAGUUGUGUGUCAACCUUGAAGGAGGGUACAAGUGCCAGUGCGAGGAGGGAUUUCAGCUUGACCCGCACACGAAAGCAUGUAAAGCGGUGGGGUCCAUUGCGUAUUUGUUUUUCACAAACAGACAUGAAGUGCGGAAGAUGACCCUUGAUCGCAGCGAAUAUACGUCACUGAUCCCUAAUCUUAGGAAUGUCGUGGCCCUUGACACGGAGGUAGCAUCAAAUAGAAUCUACUGGUCCGACCUCUCACAGAGAAUGAUCUGUUCAACACAGUUGGAUCGGGCGCACGGGGUGUCGUCGUACGAUACGGUAAUUAGCCGCGACAUCCAGGCGCCAGACGGACUCGCGGUCGACUGGAUCCAUAGCAACAUCUACUGGACAGACUCCGUGUUGGGAACCGUAUCCGUAGCUGACACAAAGGGAGUGAAGCGGAAAACUCUUUUUAGAGAGAACGGCAGCAAACCGAGAGCAAUCGUGGUCGAUCCGGUGCAUGGAUUCAUGUAUUGGACCGAUUGGGGAACGCCAGCCAAAAUCAAGAAAGGCGGUUUGAAUGGGGUCGACAUCUACUCGCUGGUGACUGAGAAUAUUCAGUGGCCAAACGGGAUCACCUUGGACUUGUUGUCGGGGAGGUUGUAUUGGGUGGACUCAAAGCUCCACUCGAUCAGCUCGAUCGACGUGAACGGCGGAAAUAGGAAAACUAUUCUCGAAGAUGAGAAAAGACUGGCCCACCCCUUCUCGCUCGCGGUGUUCGAGGACAAAGUAUUUUGGACAGACAUCAUCAACGAAGCGAUCUUUUCAGCCAACCGCCUGACAGGGUCGGAUGUCAAUCUCUUGGCCGAAAACCUUCUGAGCCCGGAAGAUAUGGUCUUGUUUCACAAUUUGACCCAACCCAGAGGUGUGAAUUGGUGCGAACGGACGACAUUGUCGAACGGAGGUUGCCAGUAUCUCUGUCUCCCUGCACCCCAGAUUAAUCCCCAUUCACCCAAGUUCACGUGUGCGUGCCCAGACGGAAUGCUUCUUGCGAGGGACAUGAGAUCCUGUCUCACCGAAGCGGAAGCGGCAGUGGCCACACAAGAGACUUCGACUGUCCGCCUUAAAGUGUCCUCGACGGCGGUCCGAACUCAGCAUACGACCACACGACCCGUGCCCGAUACCUCGCGGUUGCCCGGAGCAACACCGGGGUUGACGACAGUAGAAAUCGUAACCAUGAGCCACCAGGCACUUGGAGAUGUCGCAGGCAGAGGCAAUGAGAAGAAACCCAGCUCGGUCAGAGCCCUCAGCAUCGUGCUGCCUAUUGUGCUGCUUGUGUUUCUCUGUUUGGGUGUGUUCUUGUUGUGGAAGAACUGGCGCCUUAAGAAUAUCAACUCGAUUAACUUCGAUAAUCCGGUAUACCAGAAAACCACAGAGGAUGAAGUGCAUAUUUGUCACAACCAAGAUGGCUAUUCGUACCCGUCCAGGCAAAUGGUAUCACUUGAGGACGACGUGGCCUGAUAAGCUGCCUUCUGCGGGGCUUGCCUUCUGGCCAUGCCCUUCUUCUCUCCCUUGCACCUGUACCUCUUGGUCUUUGAAUAAAGCCUGAGUAGGAAG mCherryGGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCAC 43 mRNA CAUGGUAUCCAAGGGGGAGGAGGACAACAUGGCGAUCAUCAAGGAGU sequenceUCAUGCGAUUCAAGGUGCACAUGGAAGGUUCGGUCAACGGACACGAAUUUGAAAUCGAAGGAGAGGGUGAAGGAAGGCCCUAUGAAGGGACACAGACCGCGAAACUCAAGGUCACGAAAGGGGGACCACUUCCUUUCGCCUGGGACAUUCUUUCGCCCCAGUUUAUGUACGGGUCCAAAGCAUAUGUGAAGCAUCCCGCCGAUAUUCCUGACUAUCUGAAACUCAGCUUUCCCGAGGGAUUCAAGUGGGAGCGGGUCAUGAACUUUGAGGACGGGGGUGUAGUCACCGUAACCCAAGACUCAAGCCUCCAAGACGGCGAGUUCAUCUACAAGGUCAAACUGCGGGGGACUAACUUUCCGUCGGAUGGGCCGGUGAUGCAGAAGAAAACGAUGGGAUGGGAAGCGUCAUCGGAGAGGAUGUACCCAGAAGAUGGUGCAUUGAAGGGGGAGAUCAAGCAGAGACUGAAGUUGAAAGAUGGGGGACAUUAUGAUGCCGAGGUGAAAACGACAUACAAAGCGAAAAAGCCGGUGCAGCUUCCCGGAGCGUAUAAUGUGAAUAUCAAGUUGGAUAUUACUUCACACAAUGAGGACUACACAAUUGUCGAACAGUACGAACGCGCUGAGGGUAGACACUCGACGGGAGGCAUGGACGAGUUGUACAAAUGAUAAGCUGCCUUCUGCGGGGCUUGCCUUCUGGCCAUGCCCUUCUUCUCUCCCUUGCACCUGUACCUCUUGGUCUUUGAAUAAAGCCUGA GUAGGAAG LuciferaseGGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCAC 44 mRNA CAUGGAAGAUGCGAAGAACAUCAAGAAGGGACCUGCCCCGUUUUACCC sequenceUUUGGAGGACGGUACAGCAGGAGAACAGCUCCACAAGGCGAUGAAACGCUACGCCCUGGUCCCCGGAACGAUUGCGUUUACCGAUGCACAUAUUGAGGUAGACAUCACAUACGCAGAAUACUUCGAAAUGUCGGUGAGGCUGGCGGAAGCGAUGAAGAGAUAUGGUCUUAACACUAAUCACCGCAUCGUGGUGUGUUCGGAGAACUCAUUGCAGUUUUUCAUGCCGGUCCUUGGAGCACUUUUCAUCGGGGUCGCAGUCGCGCCAGCGAACGACAUCUACAAUGAGCGGGAACUCUUGAAUAGCAUGGGAAUCUCCCAGCCGACGGUCGUGUUUGUCUCCAAAAAGGGGCUGCAGAAAAUCCUCAACGUGCAGAAGAAGCUCCCCAUUAUUCAAAAGAUCAUCAUUAUGGAUAGCAAGACAGAUUACCAAGGGUUCCAGUCGAUGUAUACCUUUGUGACAUCGCAUUUGCCGCCAGGGUUUAACGAGUAUGACUUCGUCCCCGAGUCAUUUGACAGAGAUAAAACCAUCGCGCUGAUUAUGAAUUCCUCGGGUAGCACCGGUUUGCCAAAGGGGGUGGCGUUGCCCCACCGCACUGCUUGUGUGCGGUUCUCGCACGCUAGGGAUCCUAUCUUUGGUAAUCAGAUCAUUCCCGACACAGCAAUCCUGUCCGUGGUACCUUUUCAUCACGGUUUUGGCAUGUUCACGACUCUCGGCUAUUUGAUUUGCGGUUUCAGGGUCGUACUUAUGUAUCGGUUCGAGGAAGAACUGUUUUUGAGAUCCUUGCAAGAUUACAAGAUCCAGUCGGCCCUCCUUGUGCCAACGCUUUUCUCAUUCUUUGCGAAAUCGACACUUAUUGAUAAGUAUGACCUUUCCAAUCUGCAUGAGAUUGCCUCAGGGGGAGCGCCGCUUAGCAAGGAAGUCGGGGAGGCAGUGGCCAAGCGCUUCCACCUUCCCGGAAUUCGGCAGGGAUACGGGCUCACGGAGACAACAUCCGCGAUCCUUAUCACGCCCGAGGGUGACGAUAAGCCGGGAGCCGUCGGAAAAGUGGUCCCCUUCUUUGAAGCCAAGGUCGUAGACCUCGACACGGGAAAAACCCUCGGAGUGAACCAGAGGGGCGAGCUCUGCGUGAGAGGGCCGAUGAUCAUGUCAGGUUACGUGAAUAACCCUGAAGCGACGAAUGCGCUGAUCGACAAGGAUGGGUGGUUGCAUUCGGGAGACAUUGCCUAUUGGGAUGAGGAUGAGCACUUCUUUAUCGUAGAUCGACUUAAGAGCUUGAUCAAAUACAAAGGCUAUCAGGUAGCGCCUGCCGAGCUCGAGUCAAUCCUGCUCCAGCACCCCAACAUUUUCGACGCCGGAGUGGCCGGGUUGCCCGAUGACGACGCGGGUGAGCUGCCAGCGGCCGUGGUAGUCCUCGAACAUGGGAAAACAAUGACCGAAAAGGAGAUCGUGGACUACGUAGCAUCACAAGUGACGACUGCGAAGAAACUGAGGGGAGGGGUAGUCUUUGUGGACGAGGUCCCGAAAGGCUUGACUGGGAAGCUUGACGCUCGCAAAAUCCGGGAAAUCCUGAUUAAGGCAAAGAAAGGCGGGAAAAUCGCUGUCUGAUAAGCUGCCUUCUGCGGGGCUUGCCUUCUGGCCAUGCCCUUCUUCUCUCCCUUGCACCUGUACCUCUUGGUCUUUGAAUAAAGCCUGAGUAGGAAG

Example 15. LDLR In Vivo Study in Mammals

Low density lipoprotein (LDL) receptor (LDLR) mRNA (mRNA sequence shownin SEQ ID NO: 42; fully modified with 5-methylcytosine andpseudouridine; 5′cap, Cap1; polyA tail of 160 nucleotides not shown inthe sequence) was complexed with Lipofectamine 2000 by mixing 8.0 μgmRNA with Dulbecco's modified Eagle's medium (DMEM) to a final volume of0.2 mL.

Lipofectamine 2000 was diluted 12.5-fold with DMEM and mixed with anequivalent volume of the diluted LDL receptor mRNA solution. The sampleswere incubated 5 minutes at room temperature and a 0.1 mL volume of thecomplexed mRNA mixture was injected into the tail vein of each of threeC57BL/6 mice. Each animal received a total dose of 2.0 μg of LDLreceptor mRNA. After 6 hours, the animals were sacrificed and thespleens were removed. Splenocytes were isolated according to standardprocedures (with no prior lysis of red blood cells) and stained withequivalent amounts of either IgG specific for the human LDL receptor ornon-immune IgG as a control.

The expression of the LDL receptors was assessed by flow cytometry withgating on the CD11b+ splenocyte population. As shown in FIG. 3, theexpression of LDL receptors in the CD11b+ splenocyte population in vivowas evident in each of three separate mice by the presence of rightwardshifted peaks that were stained with LDL receptor IgG (LDLR IgG) ascompared to cells stained with non-immune IgG (non-immune IgG).

For mice treated with Lipofectamine alone, no LDL receptor specific peakwas observed and staining was similar to that observed with non-immuneIgG.

Example 16. In Vivo Expression of LDLR in Mice

LDLR −/− mice are used to test the in vivo expression of LDLR mmRNA.LDLR mmRNA is administered to LDLR −/− mice by injection. Tissues fromthe mice are examined for LDLR expression. Western blot analysis ofmouse tissues is carried out to look for LDLR protein expression as aresult of LDLR mmRNA administration. Real time RT-PCR is carried out onmouse tissues to look for LDLR gene expression.

Example 17. Confirmation of Peptide Identity

Proteins can be evaluated using liquid chromatography-mass spectrometryin tandem with mass spectrometry (LC-MS/MS) with quantitativeLC-multiple reaction monitoring (MRM) in order to confirm the identityof the peptide.

The identity of any protein target described herein can be evaluatedusing the liquid chromatography-mass spectrometry in tandem with massspectrometry (LC-MS/MS) with quantitative LC-multiple reactionmonitoring (MRM) Assay (Biognosys AG, Schlieren Switzerland). HeLa celllysates containing protein expressed from modified mRNA are evaluatedusing LC-MS/MS with quantitative LC-MRM Assay (Biognosys, SchlierenSwitzerland) in order to confirm the identity of the peptides in thecell lysates. The identified peptide fragments are compared againstknown proteins including isoforms using methods known and/or describedin the art.

A. Sample Preparation

Protein in each sample in lysis buffer is reduced by incubation for 1hour at 37° C. with 5 mM tris(2-carboxyethyl)phosphine (TCEP).Alkylation is carried out using 10 mM iodoacetamide for 30 minutes inthe dark at room temperature. Proteins are digested to peptides usingtrypsin (sequence grade, PromegaCorporation, Madison, Wis.) at aprotease: protein ratio of 1:50. Digestion is carried out overnight at37° C. (total digestion time is 12 hours). Peptides are cleaned up formass spectrometric analysis using C18 spin columns (The Nest Group,Southborough, Mass.) according to the manufacturer's instructions.Peptides are dried down to complete dryness and resuspended in LCsolvent A (1% acetonitrile, 0.1% formic acid (FA)). All solvents areHPLC-grade from SIGMA-ALDRICH® (St. Louis, Mo.) and all chemicals, wherenot stated otherwise, are obtained from SIGMA-ALDRICH® (St. Louis, Mo.).

B. LC-MS/MS and LC-MRM

Peptides are injected to a packed C18 column (Magic AQ, 3 um particlesize, 200 A pore size, Michrom Bioresources, Inc (Auburn, Calif.); 11 cmcolumn length, 75 um inner diameter, New Objective (Woburn, Mass.)) on aProxeon Easy nLC nano-liquid chromatography system for all massspectrometric analysis. LC solvents are A: 1% acetonitrile in water with0.1% FA; B: 3% water in acetonitrile with 0.1% FA. The LC gradient forshotgun analysis is 5-35% solvent B in 120 minutes followed by 35-100%solvent B in 2 minutes and 100% solvent B for 8 minutes (total gradientlength is 130 minutes). LC-MS/MS shotgun runs for peptide discovery arecarried out on a Thermo Scientific (Thermo Fisher Scientific)(Billerica, Mass.) Q Exactive mass spectrometer equipped with a standardnano-electrospray source. The LC gradient for LC-MRM is 5-35% solvent Bin 30 minutes followed by 35-100% solvent B in 2 minutes and 100%solvent B for 8 minutes (total gradient length is 40 minutes). TheThermo Scientific (Thermo Fisher Scientific) (Billerica, Mass.) TSQVantage triple quadrupole mass spectrometer is equipped with a standardnano-electrospray source. In unscheduled MRM mode for recalibration itis operated at a dwell time of 20 ms per transition. For relativequantification of the peptides across samples, the TSQ Vantage isoperated in scheduled MRM mode with an acquisition window length of 4minutes. The LC eluent is electrosprayed at 1.9 kV and MRM analysis isperformed using a Q1 peak width of 0.7 Da. Collision energies arecalculated for the TSQ Vantage by a linear regression according to thevendor's specifications.

C. Assay Design, Data Processing and Analysis

For the generation of LC-MRM assays, the 12 most intense fragment ionsfrom LC-MS/MS analysis are measured in scheduled LC-MRM mode and datawere processed using MQUEST® (Cluetec, Karlsruhe, Germany), the scoringpart of mProphet (Reiter et al, mProphet: Automated data processing andstatistical validation for large-scale SRM experiments, Nature Methods,2011 (8), 430-435; the contents of which are herein incorporated byreference). Assays were validated manually, exact fragment intensitiesare determined and iRTs (indexed retention times) are assigned relativeto Biognosys's iRT-peptides (Escher et al. Using iRT, a normalizedretention time for more targeted measurement of peptides, Proteomics,2012 (12), 1111-1121; the contents of which are herein incorporated byreference).

For the relative quantification of the peptides across the sample seriesthe 8 most intense transitions of each assay are measured across thesample series. Data analysis is carried out using SpectroDive™(Biognosys, Schlieren Switzerland). Total peak areas are compared forthe selected peptides and a false discover rate of 0.05 is applied.Peptides with a Qvalue below 0.05 are excluded and considered notdetected in the respective sample.

Example 18. Confirmation of Peptide Identity from Modified mRNAContaining Chemical Modifications

Cell lysates containing protein produced from low density lipoproteinreceptor (LDLR) modified mRNA (mRNA sequence shown in SEQ ID NO: 42;polyA tail of approximately 140 nucleotides not shown in sequence;5′cap, Cap1; fully modified with 5-methylcytosine and pseudouridine),were evaluated using the LC-MS/MS with quantitative LC-MRM as describedin Example 17. Peptide fragments identified for the evaluated proteinsare shown in Table 8. All peptides were specific for parent protein andLDLR and HFE2 was specific for the parent protein and its isoforms. InTable 8, “Uniprot ID” refers to the protein identifier from the UniProtdatabase when the peptide fragment sequences were blasted against allreview proteins in the database. Housekeeping proteins used to evaluatethe protein in the cell lysates are shown in Table 9.

TABLE 8 Protein and Peptide Fragment Sequences Peptide  Fragment SEQ IDUniprot Protein Fragment Sequence NO ID LDLR MICSTQLDR 45 P01130,P01130-4, P01130-3, P01130-2 LDLR LAHPFSLAVFEDK 46 P01130, P01130-4,P01130-3, P01130-2 LDLR NVVALDTEVASNR 47 P01130, P01130-4, P01130-3,P01130-2 LDLR TCSQDEFR 48 P01130, P01130-4

TABLE 9 Housekeeping Proteins Peptide Fragment Peptide Fragment SEQ IDProtein Sequence NO Uniprot ID Beta-actin (ACTB) VAPEEHPVLLTEAPLNPK 49P60709 Glyceraldehyde-3- VVDLMAHMASK 50 P04406 phosphate dehydrogenase(G3P) Heat shock protein HLEINPDHPIVETLR 51 P08238 HSP 90-beta (HS90B)Heat shock protein YIDQEELNK 52 P08238 HSP 90-beta (HS90B) L-lactateDQLIYNLLK 53 P00338 dehydrogenase A chain (LDHA) L-lactateGEMMDLQHGSLFLR 54 P00338 dehydrogenase A chain (LDHA) PhosphoglycerateALESPERPFLAILGGAK 55 P00558 kinase 1 (PGK1) PhosphoglycerateLGDVYVNDAFGTAHR 56 P00558 kinase 1 (PGK1) 60S acidic ribosomalIIQLLDDYPK 57 P05388 protein P0 (RLA0)

Example 19. Detection of Low Density Lipoprotein Receptor Expression inCell Culture

A. HeLa Cell Transfection

HeLa cells were plated into 24-well dishes (Corning Life Sciences,Tewksbury, Mass.) (7.5×10⁴ cells/well) in Eagles Minimal EssentialMedium (EMEM, Life Technologies, Grand Island, N.Y.) supplemented with10% fetal calf serum (FCS, Life Technologies, Grand Island, N.Y.) and 1×glutamax reagent (Life Technologies, Grand Island, N.Y.) culturedovernight under standard cell culture conditions. Transfection solutionswere prepared for each well to be treated by combining 250 ng of lowdensity lipoprotein receptor (LDLR) modified mRNA (mRNA sequence shownin SEQ ID NO: 42; polyA tail of approximately 140 nucleotides not shownin sequence; 5′cap, Cap1) fully modified with 5-methylcytosine andpseudouridine, mCherry modified mRNA (mRNA sequence shown in SEQ ID NO:43; polyA tail of approximately 160 nucleotides not shown in sequence;5′cap, Cap1; fully modified with 5-methylcytosine and pseudouridine) orluciferase modified mRNA (mRNA sequence shown in SEQ ID NO: 44; polyAtail of approximately 160 nucleotides not shown in sequence; 5′ cap,Cap1; fully modified with 5-methylcytosine and pseudouridine) with 50 μlOpti-MEM reagent (Life Technologies, Grand Island, N.Y.) in a first tubeand 1 μl of L2000 transfection reagent (Life Technologies, Grand Island,N.Y.) in 50 μl of Opti-MEM in a second tube. After preparation, firstand second tubes were incubated at room temperature for 5 minutes beforecombining the contents of each. Combined transfection solutions wereincubated for 15 minutes at room temperature. 100 μl of transfectionsolution was then added to each well. Cells were cultured for anadditional 16 hours before continued analysis.

B. LDLR Detection by Flow Cytometry

After transfection, medium were removed from cells and 60 μl of 0.25%trypsin (Life Technologies, Grand Island, N.Y.) was added to each well.Cells were trypsinized for 2 minutes before the addition of 240 μl/wellof trypsin inhibitor (Life Technologies, Grand Island, N.Y.). Resultingcell solutions were transferred to 96 well plates (Corning LifeSciences, Tewksbury, Mass.), cells were pelleted by centrifugation(800×gravity for 5 minutes) and supernatants were discarded. Cellpellets were washed with PBS and resuspended in Foxp3Fixation/Permeabilization solution (eBioscience, San Diego, Calif.) for45 minutes. Cells were pelleted again by centrifugation (800×gravity for5 minutes) and resuspended in permeabilization buffer (eBiosciences, SanDiego, Calif.) for 10 minutes. Cells were pelleted again bycentrifugation (800×gravity for 5 minutes) and washed inpermeabilization buffer. Cells were then treated with primary antibodiesdirected toward LDLR, followed by phycoerythrin-labeled secondaryantibodies. Labeled cells were then combined with FACS buffer (PBS with1% bovine serum albumin and 0.1% sodium azide) and transferred tocluster tubes. As shown in FIG. 4, labeled cells were then analyzed byflow cytometry using a BD Accuri (BD Biosciences, San Jose, Calif.).

C. LDLR Detection by Immunofluorescence

Transfected cells were washed with PBS, and treated with fixationsolution (PBS with 4% formaldehyde) for 20 minutes at room temperature.Cells were then washed with PBS and treated withpermeabilization/blocking solution (Tris buffered saline with 5% bovineserum albumin with 0.1% Tween-20). Cells were incubated for 2 hours atroom temperature with gentle agitation before washing 3 times with PBScontaining 0.05% Tween-20. Cells were then treated with or withoutprimary antibodies (goat anti-LDLR, R&D Systems, Minneapolis, Minn.) ornormal IgG controls for 2 hours at room temperature, washed 3 times withPBS containing 0.05% Tween-20 and treated with secondary antibodysolutions containing a 1:200 dilution of donkey anti-goat IgG withfluorescent label (R&D Systems, Minneapolis, Minn.). Cells were againwashed with PBS containing 0.05% Tween-20 and examined by fluorescencemicroscopy imaging. Cells transiently expressing luciferase or mCherrywere examined by fluorescence microscopy without fluorescentimmunostaining.

Example 20. Low Density Lipoprotein Receptor (LDLR) Expression

A. In Vitro LDLR Expression

Human embryonic kidney epithelial (HEK293) cells (LGC standards GmbH,Wesel, Germany) were seeded on 6-well plates (BD Biosciences, San Jose,USA). HEK293 were seeded at a density of about 500,000 cells per well in3 mL cell culture medium. Lipofectomine alone or Lipofectaminecontaining LDLR mRNA (mRNA shown in SEQ ID NO: 42; fully modified with5-methylcytosine and 1-methylpseudouridine; 5′cap, cap 1, polyA tail ofapproximately 160 nucleotides (not shown in sequence)) or Lipofectaminecontaining a control of G-CSF mRNA (mRNA shown in SEQ ID NO: 30; fullymodified with 5-methylcytosine and pseudouridine; 5′cap, cap1; polyAtail of approximately 160 nucleotides not shown in sequence) were addeddirectly after seeding the cells at quantities of 4000, 800, 400, 40,and 4 ng of LDLR modified mRNA per well and incubated. After eighteenhours incubation at 37° C., the cells were washed, fixed and stained.G-CSF mRNA transfected cells were treated with anti-LDLR antibody andone set of LDLR transfected cells were treated with normal goat IgG ascontrols. Bound primary antibodies were detected by FACS analysisfollowing treatment with a Phycoerythrin (PE)-labeled secondaryantibody.

As shown in FIG. 5A, results of the FACS analyses shows 74.8% of allgated live cells were detected to express LDLR at the 800 ng dose ofLDLR mRNA. At the 40 ng dose of LDLR mRNA, 11.6% of all gated live cellswere detected to express LDLR. No staining was observed in LDLR mRNAtreated cells stained with the control nonimmune IgG. No LDLR positivecells were detected in cells transfected with G-CSF mRNA.

B. Protein Accumulation

Human embryonic kidney epithelial (HEK293) cells (LGC standards GmbH,Wesel, Germany) were seeded on 6-well plates (BD Biosciences, San Jose,USA). HEK293 were seeded at a density of about 500,000 cells per well in3 mL cell culture medium. Lipofectamine or Lipofectamine containing LDLRmRNA (mRNA shown in SEQ ID NO: 42; fully modified with 5-methylcytosineand 1-methylpseudouridine; 5′cap, cap 1, polyA tail of approximately 160nucleotides (not shown in sequence)) or Lipofectamine containing acontrol of G-CSF mRNA (mRNA shown in SEQ ID NO: 30; fully modified with5-methylcytosine and pseudouridine; 5′ cap, cap 1; polyA tail ofapproximately 160 nucleotides not shown in sequence) were added directlyafter seeding the cells per well and incubated. Fifteen hours later thetransfection media was replaced with complete media. Transfected cellswere harvested at 0, 2, 4, 8, 24, 48, and 72 hours after mediareplacement. Transfected cells were treated with anti-LDLR antibodyconjugated to Phycoerythrin (PE) and one set of LDLR transfected cellswere treated with normal goat IgG conjugated to PE as controls. Boundprimary antibodies were detected by FACS analysis as described above.

As shown in FIG. 5B, the FACS analysis shows ˜65% of all gated livecells were detected to express LDLR at the 0.0-h time point (15.0-hafter transfection) after washing away the transfection media. Thepercent positive cells declined with time at 37° C., such that by 24hpost-removal of the transfection media, LDLR was not detected.

C. BODIPY®-Labeled LDLR

To evaluate whether the expressed LDLR were functional, BODIPY®-labeledLDL (Life Technologies, Woburn, Mass.) was used. HEK293 cells weretransfected overnight with either LDLR modified mRNA (mRNA shown in SEQID NO: 42; fully modified with 5-methylcytosine and1-methylpseudouridine; 5′ cap, cap 1, polyA tail of approximately 16nucleotides (not shown in sequence)) or G-CSF modified mRNA (mRNA shownin SEQ ID NO: 30; fully modified with 5-methylcytosine andpseudouridine; 5′cap, cap1; polyA tail of approximately 160 nucleotidesnot shown in sequence), the cells were washed and incubated withincreasing amounts of BODIPY-LDL. Following incubation for 1.0-h at 37°C., the cells were washed and the binding of BODIPY-LDL was assessed byFACS. Binding of BODIPY-LDL to LDLR mRNA transfected cells was highaffinity (Kd ˜60 ng/mL) and saturable as shown in FIG. 5C. No bindingwas observed, in contrast, to cells transfected with G-CSF modifiedmRNA.

To evaluate the LDL binding specificity it was investigated whetherLDL-BODIPY binding signal could be reduced by competition with unlabeledLDL. HEK293 cells were transfected overnight with LDLR and G-CSF mRNA asa control. 0.5 ug/mL of LDL-BODIPY was added simultaneously to thetransfected cells with 0.01, 0.1, 0.5, 1.0, 10, 100 or 500 ug/mL ofunlabeled BODIPY. The percent of live gated transfected cells detectedas positive through flow cytometry for labeled LDL are shown in Table10. LDL-BODIPY signal was progressively reduced as more unlabeled LDLwas added.

TABLE 10 Percent labeled LDL staining Unlabeled LDL Labeled cellsdetected concentration (ug/mL) (%) 0 100 0.01 97.7 0.1 59.0 0.5 64.1 176.0 10 48.4 100 3.2 500 0.9

In competition studies, binding of BODIPY-LDL could be reduced in adose-dependent manner by unlabeled LDL (FIG. 5D). These data show thatbinding of BODIPY-LDL to cells expressing LDLR mRNA is saturable,specific, and of high affinity.

To assess whether expression of LDLR mRNA in vivo could reduce thelevels of plasma cholesterol, LDLR knock-out mice (Jackson Laboratories,Bar harbor, Maine) were treated by either a single 0.1 mL intravenousinjection of 2.0 μg of LDLR mRNA in Lipofectamine 2000 or were injectedwith Lipofectamine 2000 alone (shown as “Negative” in FIG. 5E). After24.0-h, serum was isolated from each mouse and fractionated by fastprotein liquid chromatography (FPLC) by size exclusion chromatography.As a positive control the active protein human growth hormone (AbcamCat# ab116162) was also used (shown as “Growth Hormone” in FIG. 5E). Thetotal cholesterol content of each fraction is shown in FIG. 5E. SDS-PAGEanalysis showed that apo B containing lipoproteins (VLDL, IDL and LDL)were confined to fractions 3-5. The average total cholesterol offractions 3 through 6 was 416.1 ug for the negative control, 409.0 ugfor the positive control (growth hormone) and 321.3 ug for the miceadministered mRNA encoding LDLR. Relative to injection of vehicle,treatment of LDLR knockout mice with a single injection of LDLR mRNAreduced the cholesterol content in the VLDL+IDL+LDL fractions byapproximately 20%. These data show that expression of LDLR mRNA in LDLRknock-out mice can reduce the serum levels of cholesterol-richlipoproteins.

Example 21. Confirmation of Peptide Identity from Modified mRNAContaining Chemical Modifications

Cell lysates containing protein produced from low density lipoproteinreceptor (LDLR) modified mRNA (mRNA sequence shown in SEQ ID NO: 42;polyA tail of approximately 140 nucleotides not shown in sequence;5′cap, Cap1) and LDLR-PCSK9-4A modified mRNA (mRNA sequence shown in SEQID NO: 12; polyA tail of approximately 140 nucleotides not shown insequence; 5′cap, Cap1) fully modified with 5-methylcytosine andpseudouridine (5mC and pU, fully modified with 5-methylcytosine and1-methylpseudouridine (5mC and 1mpU), fully modified with pseudouridine(pU), fully modified with 1-methylpseudouridine (1mpU) or where 25% ofthe uridine residues were modified with 2-thiouridine and 25% of thecytosine residues were modified with 5-methylcytosine (s2U and 5mC) wereevaluated using the LC-MS/MS with quantitative LC-MRM as described inExample 17. Peptide fragments identified for the evaluated proteins areshown in Table 11.

TABLE 11 Proteins and Peptide Fragment Sequences Peptide 5mC 5mC s2UFragment and and and SEQ ID NO pU 1mpU 5mC pU 1mpU LDLR AVGSIAYLFFTN 58YES YES — YES YES R SEYTSLIPPLR 59 — YES — — YES LDLR-PCSK9-4AAVGSIAYLFFTN 58 YES YES — YES YES R IGAECLCPDGFQ 60 YES YES — — YESLVAQR TCSQDEFR 48 YES YES — — YES

Example 22. Design and Synthesis of Wild Type LDLR and PCSK9 BindingDeficient LDLR Modified mRNAs

A. LDLR Modified mRNA

Modified mRNA encoding LDLR (mRNA sequence shown in SEQ ID NO: 42; polyAtail of approximately 160 nucleotides not shown in sequence; 5′ cap, Cap1; fully modified with 1-methylpseudouridine and 5-methylpseudouridine)was synthesized as described previously. The modified mRNA product wasanalyzed with an Agilent 2100 bioanalyzer and as shown in FIG. 6, asingle band at the expected size of ˜2.8 Kb was observed.

B. PCSK9 Binding Deficient LDLR Modified mRNA

Modified mRNAs encoding human PCSK9 binding deficient LDLRs aresynthesized as described previously. The modified mRNA encodes a PCSK9binding deficient mutant LDLR either with a single amino acidsubstitution such as Y336A (SEQ ID NO. 37), E317A (SEQ ID NO. 36), N316A(SEQ ID NO. 35), L339D (SEQ ID NO. 34), or D331E (SEQ ID NO. 33) or aquadruple mutation variant with the amino acid substitutions: N316A,E317A, Y336A and D331A (SEQ ID NO. 38), where, for example, “N316A”means amino acid Asparagine at position 316 is substituted for the aminoacid Alanine. The mutated LDLR mRNAs further include chemicalmodification described herein. Confirmation of the modified mRNA productis done by methods known in the art such as bioanalyzer and peptidedigestion.

Example 23. In Vitro Expression of LDLR Modified mRNA

Human Embryonic Kidney 293 (HEK293) cells were transfected withlipofectamine alone, lipofectamine containing modified mRNA encodingLDLR (mRNA sequence shown in SEQ ID NO: 42; polyA tail of approximately160 nucleotides not shown in sequence; 5′cap, Cap1; fully modified with1-methylpseudouridine and 5-methylcytosine) or a control oflipofectamine containing modified RNA encoding G-CSF (mRNA sequenceshown in SEQ ID NO: 30; polyA tail of approximately 160 nucleotides notshown in sequence; 5′ cap, Cap 1; fully modified with1-methylpseudouridine and 5-methylcytosine). After an 18 hour incubationat 37° C., the cells were washed, fixed and stained with eitherphycoerthrin (PE)-labeled anti-human LDLR antibody (R&D Systems,Minneapolis, Minn.; Human LDL R Affinity Purified Polyclonal AbAF2148)or PE-labeled goat non-immune IgG (R&D Systems, Minneapolis, Minn.; R&DSystems Purified goat IgG R&D Systems catalog number AC-108-C).Conjugation to PE was completed with the Innova bioscienceslightning-link conjugation kit (Lightning-Link R-PE Antibody LabelingKit Novus Biologicals catalog number: 703-0010).

The expression of human LDLR was monitored by flow cytometry.Transfection with increasing amounts (4-4000 ng) of LDLR modified mRNAincreased the percent of cells that stained with the PE-labeledanti-LDLR IgG but not in the cells transfected with control of G-CSFmodified mRNA. The transfection of 800 ng of modified mRNA encoding LDLRis shown in FIG. 7. There was no positive staining detected withPE-labeled non-immune IgG in cells transfected with LDLR modified mRNA.

LDLR expression reached a peak at 8 to 24 hours post-transfection anddeclined thereafter.

Example 24. Detection of LDLR

Human Embryonic Kidney 293 (HEK293) cells were transfected withlipofectamine containing modified mRNA encoding LDLR (mRNA shown in SEQID NO: 42, polyA of approximately 160 nucleotides not shown in sequence;5′cap, Cap1; fully modified with 1-methylpseudouridine and5-methylcytosine) or a control of lipofectamine containing modified mRNAencoding G-CSF (mRNA sequence shown in SEQ ID NO: 30; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′cap, Cap1; fullymodified with 1-methylpseudouridine and 5-methylcytosine). After anincubation of 16 hours at 37° C., the cells were washed and replacedwith complete growth media. Cells were harvested and stained for LDLR at0, 2, 4 and 8 hours post transfection. As shown in FIG. 8, LDLR wasdetected in 68.1% of live cells after transfection and diminished to27.6% at 8 hours in the absence of transfection media. In FIG. 8,columns 1 and 2 were transfected with mRNA encoding LDLR and column 3was transfected with the control mRNA encoding G-CSF. Columns 1 and 3were stained with phycoerthrin (PE)-labeled anti-human LDLR antibody(R&D Systems, Minneapolis, Minn.; Human LDL R Affinity PurifiedPolyclonal AbAF2148) and Column 2 was stained with goat IgG conjugatedto PE (R&D Systems, Minneapolis, Minn.; R&D Systems Purified goat IgGR&D Systems catalog number AC-108-C) as a control. Conjugation to PE wascompleted with the Innova biosciences lightning-link conjugation kit(Lightning-Link R-PE Antibody Labeling Kit Novus Biologicals catalognumber: 703-0010).

Example 25. In Vitro Expression of PCSK9 Binding Deficient LDLR ModifiedmRNAs

Modified mRNAs encoding human and mouse wild type LDLR and human PCSK9binding deficient LDLRs are synthesized as described herein. Themodified mRNAs are transfected with HEK293 cells by methods known in theart. The expression of wild type LDLR and PCSK9 binding deficient LDLRmutants are screened after transfections to determine the highestexpressing modified mRNAs.

Example 26. PCSK9 Down-Regulation of LDLR in Hep-G2 Cells

Human Hepatocellular carcinoma (Hep-G2) cells are cultured in completemedia (DMEM medium with 10% lipoprotein deficient serum (IntracelResources, Frederick, Md.)) to down regulate endogenous LDLRs. Thedown-regulation of LDLR expression by PCSK9 will be assessed by knownmethods (e.g., see Lipari et al., Furin-cleaved Proprotein ConvertaseSubtilisin/Kexin Type 9 (PCSK9) Is Active and Modulates Low DensityLipoprotein Receptor and Serum Cholesterol Levels. J Biol Chem. 2012,287(52): 43482-43491; McNutt et al. Antagonism of Secreted PCSK9Increases Low Density Lipoprotein Receptor Expression in HepG2 Cells. JBiol Chem. 2009. 284(16): 10561-10570; each of which is hereinincorporated by reference in its entirety).

One method to assess the down-regulation of LDLR expression istransfecting Hep-G2 cells with wild type LDLR modified mRNA or PCSK9binding deficient LDLR modified mRNAs as described herein. The Hep-G2cells are cultured in complete media to down-regulate endogenous LDLRsprior to transfection with LDLR modified mRNA. After 24 hour incubationat 37° C., the turnover of LDLR from transfection with modified mRNAencoding wild type LDLR or PCSK9 binding deficient LDLR is assessed inthe presence and absence of exogenous PCSK9 (R &D Systems, Minneapolis,Minn.) by western blot analysis of cell lysates (PROTEIN SIMPLE™, SantaClara, Calif.) and by flow cytometry (e.g., FACS sorting). The modifiedmRNA encoding the most PCSK9-insensitive LDLR is determined.

Example 27. Liver Cell Transducing Formulations

Lipid nanoparticles (LNPs) are formulated using methods known in theart, described herein and/or described in PCT/US2012/069610 entitled“Modified Nucleoside, Nucleotide, and Nucleic Acid Composition,” hereinincorporated by reference in its entirety. The LNPs used herein cancomprise the ionizable lipid DLin-KC2-DMA or the cationic lipid C12-200.

Modified mRNA encoding luciferase (e.g., SEQ ID NO: 44; polyA tail of atleast 140 nucleotides not shown in sequence; 5′cap, Cap1; modified withat least one chemical modification described herein) is formulated inLNPs comprising DLin-KC2-DMA or C12-200. The formulated luciferase isadministered to wild type mice and LDLR deficient mice. The expressionof luciferase in the liver cells of the wild type and LDLR deficientmice is measured, using methods known in the art or described herein, atpredetermined intervals after administration of the modified mRNA.Twenty minutes prior to imaging, mice are injected intraperitoneallywith a D-luciferin solution at 150 mg/kg. Animals are anesthetized andimages are acquired with an IVIS lumina II imaging system (Perkin Elmer,Waltham, Mass.). Bioluminescenes are measured as total flux(photons/second).

Example 28. In Vivo Expression of LDLR in Mice

LDLR −/− mice are used to test the in vivo expression of modified mRNAsencoding the wild type human or murine LDLR or encoding PCSK9 bindingdeficient human or murine LDLRs (collectively, “LDLR modified mRNAs”).LDLR modified mRNAs formulated in lipid nanoparticles are administeredto LDLR −/− mice through 0.1 ml intravenous injections containingincreasing doses of between 0.005-0.5 mg/kg (e.g. 0.005 mg/kg, 0.010mg/kg, 0.015 mg/kg, 0.020 mg/kg, 0.030 mg/kg, 0.040 mg/kg, 0.050 mg/kg,0.1 mg/kg, 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg and 0.5 mg/kg) of the LDLRmodified mRNAs. Mice are sacrificed at various times, such as between 2hours and 96 hours (e.g., 2 hr, 2.5 hr, 3 hr, 3.5 hr, 4 hr, 5 hr, 6 hr,7 hr, 8 hr, 9 hr, 10 hr, 12 hr, 24 hr, 48 hr, 72 hr and 96 hr) after theinjection. The livers are excised and the cell lysates and livers areprepared for analysis. The LDLR protein expression and drug levelchanges in mRNA transcript are measured by western blot analysis usingan anti-LDLR antibody and the expression of modified LDLR mRNA in mousetissues is analyzed by real time RT-PCR. Serum is collected at varioustimepoints after injection of the modified mRNA and analyzed forcytokine panels using a mouse LUMINEX® panel. The remainder of the serawill be fractionated by FPLC for assay of VLDL+IDL+LDL cholesterol (seethe method described in Garber et al. A sensitive and convenient methodfor lipoprotein profile analysis of individual mouse plasma samples.Journal of Lipid Research. 2000. 14: 1020-1026; herein incorporated byreference in its entirety).

In a further study, the LDLR −/− mice are administered more than oncewith modified mRNAs encoding the wild type human or murine LDLR orencoding PCSK9 binding deficient human or murine LDLRs. Serum iscollected at various timepoints after injection of the modified mRNA andanalyzed for cytokine panels using a mouse LUMINEX® panel and assayedfor VLDL+IDL+LDL cholesterol. The mice are also sacrificed and thelivers are excised and cell lysates are prepared for analysis.

Example 29. In Vivo Expression of LDLR in the Watanabe (WHHL) Rabbit

Watanabe (WHHL) rabbits are used to test the in vivo expression ofmodified mRNAs encoding the wild type human LDLR or encoding PCSK9binding deficient human LDLRs (“LDLR modified mRNAs”). LDLR modifiedmRNAs formulated in lipid nanoparticles are administered throughinjections containing 0.005-0.5 mg/kg (e.g., 0.005 mg/kg, 0.010 mg/kg,0.015 mg/kg, 0.020 mg/kg, 0.030 mg/kg, 0.040 mg/kg, 0.050 mg/kg, 0.1mg/kg, 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg and 0.5 mg/kg) of the LDLRmodified mRNAs to the rabbits. The WHHL rabbits are sacrificed between 2hours and 96 hours (e.g., 2 hr, 2.5 hr, 3 hr, 3.5 hr, 4 hr, 5 hr, 6 hr,7 hr, 8 hr, 9 hr, 10 hr, 12 hr, 24 hr, 48 hr, 72 hr and 96 hr) after theinjection and the livers are excised and cell lysates are prepared foranalysis. The LDLR protein expression and changes in mRNA transcript ismeasured in the cell lysates by western blot analysis using an anti-LDLRantibody and the expression of LDLR in rabbit tissues is analyzed byreal time RT-PCR. Serum is collected at various timepoints afterinjection of the modified mRNA and analyzed for cytokine panels andassayed for VLDL+IDL+LDL cholesterol.

Example 30. In Vivo Expression of LDLR in the LDLR Deficient Pigs

LDLR deficient pigs (Exemplar Genetics, Sioux Center, Iowa) are used totest the in vivo expression of modified mRNAs encoding the wild typehuman LDLR or encoding PCSK9 binding deficient human LDLRs (“LDLRmodified mRNAs”). LDLR modified mRNAs formulated in lipid nanoparticlesare administered through injections containing 0.005-0.5 mg/kg (e.g.,0.005 mg/kg, 0.010 mg/kg, 0.015 mg/kg, 0.020 mg/kg, 0.030 mg/kg, 0.040mg/kg, 0.050 mg/kg, 0.1 mg/kg, 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg and 0.5mg/kg) of the LDLR modified mRNAs to the LDLR deficient pigs. The pigsare sacrificed between 2 hours and 96 hours (e.g., 2 hr, 2.5 hr, 3 hr,3.5 hr, 4 hr, 5 hr, 6 hr, 7 hr, 8 hr, 9 hr, 10 hr, 12 hr, 24 hr, 48 hr,72 hr and 96 hr) after the injection and the livers are excised and celllysates are prepared for analysis. The LDLR protein expression andchanges in mRNA transcript is measured in the cell lysates by westernblot analysis using an anti-LDLR antibody and the expression of LDLR inpig tissues is analyzed by real time RT-PCR. Serum is collected atvarious timepoints after injection of the modified mRNA and analyzed forcytokine panels and assayed for VLDL+IDL+LDL cholesterol.

Example 31. In Vivo Expression of LDLR in LDLR Deficient Rhesus Monkeys

LDLR deficient rhesus monkeys (Southwest National Primate ResearchCenter, San Antonio, Tex.) are used to test the in vivo expression ofmodified mRNAs encoding the wild type human LDLR or encoding PCSK9binding deficient human LDLRs (“LDLR modified mRNAs”). LDLR modifiedmRNAs formulated in lipid nanoparticles are administered throughinjections containing 0.005-0.5 mg/kg (e.g., 0.005 mg/kg, 0.010 mg/kg,0.015 mg/kg, 0.020 mg/kg, 0.030 mg/kg, 0.040 mg/kg, 0.050 mg/kg, 0.1mg/kg, 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg and 0.5 mg/kg) of the LDLRmodified mRNAs to the LDLR deficient monkeys. The monkeys are sacrificedbetween 2 hours and 96 hours (e.g., 2 hr, 2.5 hr, 3 hr, 3.5 hr, 4 hr, 5hr, 6 hr, 7 hr, 8 hr, 9 hr, 10 hr, 12 hr, 24 hr, 48 hr, 72 hr and 96 hr)after the injection and the livers are excised and cell lysates areprepared for analysis. The LDLR protein expression and changes in mRNAtranscript is measured in the cell lysates by western blot analysisusing an anti-LDLR antibody and the expression of LDLR in monkey tissuesis analyzed by real time RT-PCR. Serum is collected at varioustimepoints after injection of the modified mRNA and analyzed forcytokine panels and assayed for VLDL+IDL+LDL cholesterol.

Example 32. Multi-Dose Studies

Studies utilizing multiple doses are designed and performed usingLDLR−/− mice. The mice are injected intravenously eight times (twice aweek) over 28 days with 0.5 mg/kg, 0.05 mg/kg, 0.005 mg/kg or 0.0005mg/kg of modified LDLR mRNA encoding human or mouse wild type LDLR orencoding PCSK9 binding deficient human LDLR, formulated in a lipidnanoparticle. The LDLR protein expression and changes in mRNA transcriptis measured in the cell lysates by western blot analysis using ananti-LDLR antibody and the expression of LDLR in tissues is analyzed byreal time RT-PCR. Sera are collected during pre-determined timeintervals and analyzed for cytokines panel and assay for VLDL+IDL+LDLcholesterol as described herein.

Example 33. Total Cholesterol in Wild Type and LDLR Knock Out Mice

Serum from three wild type mice (C57BL/6J) and three LDLR knock out mice(B6. 129S7-ldlrtm1Her/J, Jackson Laboratories, Bar Harbor, Me.) werecollected and fractionized by FPLC. Absorbance of eluted serum fractionsfrom wild type and LDLR knock out mice were measured at 260 and 280 nm.Each fraction was analyzed for total cholesterol by the Wako cholesterolE enzymatic colometric method (Wako, Richmond, Va.). As shown in FIG.9A, absorbance profiles showed three distinct protein peaks in bothmouse strains (FIG. 9A). In The first peak, spanning fractions 3 through6 in FIG. 9B, tested highest for cholesterol in LDLR knockout mice. Thesecond peak, spanning fractions 7 through 12 in FIG. 9B, tested highestfor total cholesterol in wild type mice. The third peak, spanningfractions 14 through 18 in FIG. 9B, tested low or negative forcholesterol in both mouse strains.

Example 34. Expression of Wild-Type LDLR and PCSK9 Binding-DeficientVariants

Human embryonic kidney epithelial (HEK293) cells (LGC standards GmbH,Wesel, Germany) are seeded on 48-well plates (BD Biosciences, San Jose,USA). HEK293 are seeded at a density of about 60,000 cells per well in0.2 mL cell culture medium. Formulations containing 1 uL lipofectamineand 150 ng of wild type (WT) LDLR mRNA (mRNA sequence shown in SEQ IDNO: 42; polyA tail of approximately 160 nucleotides not shown insequence; 5′cap, Cap1; fully modified with 5-methylcytosine and1-methylpseudouridine) or 150 ng of an LDLR sequence variant mRNA or acontrol of G-CSF mRNA (mRNA shown in SEQ ID NO: 30; fully modified with5-methylcytosine and pseudouridine; 5′cap, cap1; polyA tail ofapproximately 160 nucleotides not shown in sequence) are added directlyafter seeding the cells at quantities of 60,000 per well and incubated.

The LDLR mRNA sequence variants prepared and tested include: a fouramino acid substitution variant (4A, N316A, E317A, D331A, and Y336A)(mRNA sequence shown in SEQ ID NO: 12; polyA tail of approximately 160nucleotides not shown in sequence; 5′ cap, Cap1; fully modified with5-methylcytosine and 1-methylpseudouridine), or the following singleamino acid substitution variants Y336A (mRNA sequence shown in SEQ IDNO: 11; polyA tail of approximately 160 nucleotides not shown insequence; 5′cap, Cap1; fully modified with 5-methylcytosine and1-methylpseudouridine), E317A (mRNA sequence shown in SEQ ID NO: 10;polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1; fully modified with 5-methylcytosine and1-methylpseudouridine), N316A (mRNA sequence shown in SEQ ID NO: 9;polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1; fully modified with 5-methylcytosine and1-methylpseudouridine), L339D (mRNA sequence shown in SEQ ID NO: 8;polyA tail of approximately 160 nucleotides not shown in sequence;5′cap, Cap1; fully modified with 5-methylcytosine and1-methylpseudouridine), D331E (mRNA sequence shown in SEQ ID NO: 7;polyA tail of approximately 160 nucleotides not shown in sequence;5′cap, Cap1; fully modified with 5-methylcytosine and1-methylpseudouridine).

As controls G-CSF mRNA transfected cells were treated with anti-LDLRantibody (R&D Systems, Minneapolis, Minn.; Human LDL R Affinity PurifiedPolyclonal AbAF2148) and one set of LDLR transfected cells were treatedwith normal goat IgG (R&D Systems, Minneapolis, Minn.; Purified goat IgGR&D Systems catalog number AC-108-C). Bound primary antibodies weredetected by FACS analysis following treatment with a Phycoerythrin(PE)-labeled antibody (R&D Systems, Minneapolis, Minn.). Conjugation toPE was completed with the Innova biosciences lightning-link conjugationkit (Lightning-Link R-PE Antibody Labeling Kit Novus Biologicals catalognumber: 703-0010).

As shown in FIG. 10, the FACS analysis shows 32% of all gated live cellswere detected to express LDLR at the 150 ng dose of wild type LDLR mRNA(WT in FIG. 10). Similarly, for cells transfected with the LDLR mRNAvariants, between 12-33% of all gated live cells were detected toexpress LDLR at the 150 ng dose. No staining was observed in LDLR mRNAtreated cells stained with the control non-immune IgG (LDLR WT transfectIsotype stain) and no LDLR positive cells were detected in cellstransfected with G-CSF mRNA (GCSF transfect LDLR stain).

Example 35. Down-Modulation of LDLR by Exogenous PCSK9

Human embryonic kidney epithelial (HEK293) cells (LGC standards GmbH,Wesel, Germany) are seeded on 48-well plates (BD Biosciences, San Jose,USA). HEK293 are seeded at a density of about 60,000 cells per well in0.2 mL cell culture medium. Formulations containing 1 uL oflipofectamine 2000 and 150 ng of wild type (WT) LDLR mRNA (mRNA sequenceshown in SEQ ID NO: 42; polyA tail of approximately 160 nucleotides notshown in sequence; 5′cap, Cap1; fully modified with 5-methylcytosine and1-methylpseudouridine) or 150 ng of an LDLR sequence variant mRNA or 150ng of a control of G-CSF mRNA (mRNA shown in SEQ ID NO: 30; fullymodified with 5-methylcytosine and pseudouridine; 5′cap, cap1; polyAtail of approximately 160 nucleotides not shown in sequence) are addeddirectly after seeding the cells at quantities of 800 ng per well andincubated in the presence and in the absence of exogenous human PCSK9 at60 μs/mL.

The LDLR mRNA sequence variants prepared and tested include: a fouramino acid substitution variant (4A: N316A, E317A, D331A, and Y336A)(mRNA sequence shown in SEQ ID NO: 12; polyA tail of approximately 160nucleotides not shown in sequence; 5′ cap, Cap1; fully modified with5-methylcytosine and 1-methylpseudouridine), or the following singleamino acid substitution variants Y336A(mRNA sequence shown in SEQ ID NO:11; polyA tail of approximately 160 nucleotides not shown in sequence;5′cap, Cap1; fully modified with 5-methylcytosine and1-methylpseudouridine), E317A (mRNA sequence shown in SEQ ID NO: 10;polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1; fully modified with 5-methylcytosine and1-methylpseudouridine), N316A (mRNA sequence shown in SEQ ID NO: 9;polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1; fully modified with 5-methylcytosine and1-methylpseudouridine), L339D (mRNA sequence shown in SEQ ID NO: 8;polyA tail of approximately 160 nucleotides not shown in sequence;5′cap, Cap1; fully modified with 5-methylcytosine and1-methylpseudouridine), D331E (mRNA sequence shown in SEQ ID NO: 7;polyA tail of approximately 160 nucleotides not shown in sequence;5′cap, Cap1; fully modified with 5-methylcytosine and1-methylpseudouridine).

After fifteen hours at 37° C., the transfection media were removed, thecells were washed, and were treated with anti-LDLR antibody conjugatedto Phycoerythrin (PE) (R&D Systems, Minneapolis, Minn.; Human LDL RAffinity Purified Polyclonal AbAF2148) as described above. One set ofLDLR transfected cells were treated with normal goat IgG conjugated toPE (R&D Systems, Minneapolis, Minn.; Purified goat IgG R&D Systemscatalog number AC-108-C) and another set of untransfected cells wereused as controls. Cells transfected with G-CSF modified mRNA were usedas an additional negative control. Conjugation to PE was completed withthe Innova biosciences lightning-link conjugation kit (Lightning-LinkR-PE Antibody Labeling Kit Novus Biologicals catalog number: 703-0010).Bound primary antibodies were detected by flow cytometry as describedabove.

As shown in FIG. 11, the FACS analysis showed that cell surface LDLRexpression in cells transfected with wild-type LDLR mRNA was 51.6% ofgated live cells. This value decreased to 21.5% of gated live cells whenexogenous PCSK9 was added to the media during cell transfection. Incontrast, each of the LDLR mRNA variants with substitutions in the PCSK9binding domain showed less sensitivity to down-modulation by exogenousPCSK9 (Table 12) with values of percent reduction ranging from −8.1% to29% (compared to 58% reduction observed with wild-type LDLR.

TABLE 12 Down-Modulation of Cell Surface LDLR Expression by ExogenousPCSK9 Percent Reduction % of Cells LDLR Positive in LDLR Expression LDLRmRNA PCSK9 Absent PCSK9 Present (%) WT 51.6 21.5 58.3 N316A, E317A, 65.163.3 2.8 D331A, Y336A Y336A 73.1 52.1 28.7 E317A 71.8 51.9 27.7 N316A60.8 65.7 −8.1 L339D 69.4 60.9 12.2 D331E 69.4 60.9 0.8 G-CSF negative1.06 0.76 NA control

Example 36. Functionality of LDLR Expressed by Variant LDLR mRNA

To evaluate whether the expressed LDLR were functional, BODIPY-labeledLDL was used. 60,000 HEK293 cells per well were transfected overnightwith formulations containing 1 uL lipofectamine 2000 and 150 ng encodingwild type (WT) LDLR mRNA (mRNA sequence shown in SEQ ID NO: 42; polyAtail of approximately 160 nucleotides not shown in sequence; 5′cap,Cap1; fully modified with 5-methylcytosine and 1-methylpseudouridine) or150 ng of an LDLR variant mRNA or 150 of a control of G-CSF mRNA (mRNAshown in SEQ ID NO: 30; fully modified with 5-methylcytosine andpseudouridine; 5′cap, cap1; polyA tail of approximately 160 nucleotidesnot shown in sequence).

The LDLR variant mRNA sequences prepared and tested include: a fouramino acid substitution variant (4A: N316A, E317A, D331A, and Y336A)(mRNA sequence shown in SEQ ID NO: 12; polyA tail of approximately 160nucleotides not shown in sequence; 5′ cap, Cap1; fully modified with5-methylcytosine and 1-methylpseudouridine), or the following singleamino acid substitution variants Y336A(mRNA sequence shown in SEQ ID NO:11; polyA tail of approximately 160 nucleotides not shown in sequence;5′cap, Cap1; fully modified with 5-methylcytosine and1-methylpseudouridine), E317A (mRNA sequence shown in SEQ ID NO: 10;polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1; fully modified with 5-methylcytosine and1-methylpseudouridine), N316A (mRNA sequence shown in SEQ ID NO: 9;polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1; fully modified with 5-methylcytosine and1-methylpseudouridine), L339D (mRNA sequence shown in SEQ ID NO: 8;polyA tail of approximately 160 nucleotides not shown in sequence;5′cap, Cap1; fully modified with 5-methylcytosine and1-methylpseudouridine), D331E (mRNA sequence shown in SEQ ID NO: 7;polyA tail of approximately 160 nucleotides not shown in sequence;5′cap, Cap1; fully modified with 5-methylcytosine and1-methylpseudouridine).

The cells were washed and incubated with increasing amounts (0 ug/ml,0.1 ug/ml, 1.0 ug/ml, 10 ug/ml or 50 ug/ml) of BODIPY-LDL. Followingincubation for 1 hour at 37° C., the cells were washed and the cellassociated BODIPY-LDL was assessed by flow cytometry. The contour plotsare shown in FIG. 12A. For wild-type (WT) and LDLR PCSK9 bindingvariants, the binding of BODIPY-LDL to LDLR mRNA transfected cells wasevident as a right-ward shift in the gated cell population thatincreased with BODIPY-LDL concentration. A similar rightward shift inthe gated cell population was seen for cells transfected with each ofthe LDLR mRNA constructs. As is shown in FIG. 12B, half-maximal cellassociation was the same for BODIPY-LDL binding to cells transfectedwith either wild type or the PCSK9 binding-deficient LDLR mRNAs. Foreach construct, BODIPY-LDL binding was of high affinity (half-maximalbinding between 0.6-0.7 ng/mL) and saturable. No BODIPY-LDL binding wasobserved for cells transfected with G-CSF mRNA.

Example 37. Evaluate of Half-Life on Cell Surface LDLR

To assess whether exogenous PCSK9 can modulate the apparent half-life ofcell surface LDL receptors in cells transfected with LDLR mRNA, HEK293cells were plated on 24 well plates, 130,000 cells per well, transfectedwith LDLR mRNA and incubated for 14 hours as described above. The cellmonolayers were washed and fresh media with PCSK9 (60 μg/mL) or withoutPCSK9 was added. After various times of incubation at 37° C., cellsurface LDLR expression was monitored by flow cytometry using anti-LDLRantibodies as described above. As is shown in FIG. 13A, cell surfaceLDLR in cells transfected with 300 ng of wild-type LDLR mRNA (mRNAsequence shown in SEQ ID NO: 42; polyA tail of approximately 160nucleotides not shown in sequence; 5′cap, Cap1; fully modified with5-methylcytosine and 1-methylpseudouridine) decreased in atime-dependent manner with an apparent half-life of approximately 13hours. The addition of PCSK9 to the media of cells transfected withwild-type LDLR mRNA decreased the apparent half-life of cell surfaceLDLR to about 4 hours. In FIG. 13A, “*” represents a significantdifference by statistical test.

In contrast, the addition of PCSK9 to the media of cells transfectedwith 300 ng of PCSK9 binding-deficient LDLR mRNA produced little or nochange in the apparent half-life of cell surface LDLR (FIGS. 13B-13G).The PCSK9 binding-deficient LDLR mRNA sequences prepared and testedinclude: a four amino acid substitution variant (4A: N316A, E317A,D331A, and Y336A) (mRNA sequence shown in SEQ ID NO: 12; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′ cap, Cap1; fullymodified with 5-methylcytosine and 1-methylpseudouridine), or thefollowing single amino acid substitution variants Y336A(mRNA sequenceshown in SEQ ID NO: 11; polyA tail of approximately 160 nucleotides notshown in sequence; 5′cap, Cap1; fully modified with 5-methylcytosine and1-methylpseudouridine), E317A (mRNA sequence shown in SEQ ID NO: 10;polyA tail of approximately 160 nucleotides not shown in sequence;5′cap, Cap1; fully modified with 5-methylcytosine and1-methylpseudouridine), N316A (mRNA sequence shown in SEQ ID NO: 9;polyA tail of approximately 160 nucleotides not shown in sequence;5′cap, Cap1; fully modified with 5-methylcytosine and1-methylpseudouridine), L339D (mRNA sequence shown in SEQ ID NO: 8;polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1; fully modified with 5-methylcytosine and1-methylpseudouridine), D331E (mRNA sequence shown in SEQ ID NO: 7;polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1; fully modified with 5-methylcytosine and1-methylpseudouridine).

These data in FIGS. 13B-13G show that cells transfected with LDLR mRNAencoding LDLR with mutations in the PCSK9 binding site fail to respondto exogenous PCSK9, suggesting that these binding variants may have alonger half-life than wild-type LDLR in vivo and be useful for treatingpatients with hypercholesterolemia.

Example 38. Effect of Increasing PCSK9 Amount on Cell Surface LDLR

To evaluate the down-modulation of cell surface LDLR expression byincreasing amounts of PCSK9 added to the complete cell media, MEM(GlutaMAX, Life Science Catalog#41090-036) supplemented with 10% fetalbovine serum. HEK293 cells were plated at 300,000 cells per well,incubated for 6 hours, and transfected for 15 hours with 300 ng ofeither wild-type LDLR mRNA (mRNA sequence shown in SEQ ID NO: 42; polyAtail of approximately 160 nucleotides not shown in sequence; 5′cap,Cap1; fully modified with 5-methylcytosine and 1-methylpseudouridine) oran LDLR mRNA encoding a PCSK9 binding variant. The PCSK9 binding variantmRNA sequences prepared and tested include the single amino acidsubstitution variants of N316A (mRNA sequence shown in SEQ ID NO: 9;polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap 1; fully modified with 5-methylcytosine and1-methylpseudouridine), and D331E (mRNA sequence shown in SEQ ID NO: 7;polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1; fully modified with 5-methylcytosine and1-methylpseudouridine). A control of mRNA encoding UGT1A1 (mRNA sequenceshown in SEQ ID NO: 61; polyA tail of approximately 160 nucleotides notshown in sequence; 5′cap, Cap1; fully modified with 5-methylcytosine and1-methylpseudouridine) was also used.

After 15 hours of incubation, the cell monolayers were washed and eitherbuffer alone or buffer containing increasing amounts of PCSK9 wereadded. Cells were incubated for 5 hours and cell surface LDLR expressionwas measured by flow cytometry as described above. As is shown in FIG.14, cell surface LDL receptors in cells transfected with wild-type LDLRmRNA were decreased in a dose-dependent manner by PCSK9. Maximalreduction in LDLR was achieved at 20 μg/mL of exogenous PCSK9. Incontrast, PCSK9 had no effect on cell surface LDLR in cells transfectedwith LDLR mRNA encoding the PCSK9 binding-deficient variants N316A orD331E. No cell surface LDLR was detected in HEK293 cells transfectedwith mRNA encoding UGT1A1. These data show that LDLR expressed from LDLRmRNAs encoding mutations in the binding site for PCSK9 are insensitiveto exogenous PCSK9.

Example 39. Liver Cell Transducing Formulations of LDLR

Lipid nanoparticles (LNPs) are formulated using methods known in theart, described herein and/or described in PCT/US2012/069610 entitled“Modified Nucleoside, Nucleotide, and Nucleic Acid Composition,” hereinincorporated by reference in its entirety. The LNPs used herein cancomprise the ionizable lipid DLin-KC2-DMA or the cationic lipid C12-200.

Modified mRNA encoding LDLR or LDLR mutants (e.g., SEQ ID NOs: 7-12;polyA tail of at least 140 nucleotides not shown in sequence; 5′ cap,Cap 1; modified with at least one chemical modification describedherein) is formulated in LNPs comprising DLin-KC2-DMA or C12-200. Theformulated LDLR or LDLR mutants is administered to wild type mice andLDLR deficient mice. The expression of LDLR in the liver cells of thewild type and LDLR deficient mice is measured, using methods known inthe art or described herein, at predetermined intervals afteradministration of the modified mRNA.

Example 40. Delivery of LNP Formulated Modified mRNA

Luciferase mRNA (mRNA sequence shown in SEQ ID NO: 44; polyA tail of atleast 140 nucleotides not shown in sequence; 5′ cap, Cap1) is fullymodified with either 5-methylcytosine and pseudouridine, fully modifiedwith 5-methylcytosine and 1-methylpseudouridine, fully modified withpseudouridine, fully modified with 1-methylpseudouridine or 25% of theuridine residues are modified with 2-thiouridine and 25% of the cytosineresidues are modified with 5-methylcytosine. The luciferase mRNA is thenformulated in a lipid nanoparticle comprising the cationic lipidDLin-KC2-DMA (KC2) or C12-200. The formulated LNP in PBS or a control ofPBS alone is administered intravenously to LDLR −/− or normal mice asoutlined in Table 13. The mice are imaged at 2 hours, 8 hours, 24 hoursand 48 hours after injection. Ten minutes prior to imaging, mice areinjected intraperitoneally with a D-luciferin solution at 150 mg/kg.Animals are then anesthetized and images are acquired with an IVISLumina II imaging system (Perkin Elmer).

TABLE 13 Dosing Regimen mRNA Injection Cationic Dose dose/mouse VolumeGroup Mouse Strain Lipid (mg/kg) (mg) (mL) 1 LDLR−/− KC2 0.5 0.01 0.1 2LDLR−/− KC2 0.05 0.001 0.1 3 Normal KC2 0.5 0.01 0.1 4 Normal KC2 0.050.001 0.1 5 LDLR−/− C12-200 0.5 0.01 0.1 6 LDLR−/− C12-200 0.05 0.0010.1 7 Normal C12-200 0.5 0.01 0.1 8 Normal C12-200 0.05 0.001 0.1 9LDLR−/− none none none 0.1

Example 41. Studies of Mammals Administered UGT1A1 Modified mRNA

A. Rodents

Studies utilizing multiple doses are designed and performed using ratsand/or mice (e.g. LDLR−/− mice). The rodents are injectedintramuscularly or intravenously more than once over a period of 7 dayswith 0.5 mg/kg, 0.05 mg/kg, 0.005 mg/kg or 0.0005 mg/kg of modified LDLRmRNA encoding human, mouse or rat LDLR or its isoforms or variantsdescribed herein. The LDLR mRNA is formulated in either 5% sucrose,saline or a lipid nanoparticle. The LDLR protein expression and changesin mRNA transcript is measured in the cell lysates by western blotanalysis using an anti-LDLR antibody and the drug level and transcriptof LDLR in tissues is analyzed by real time RT-PCR. Sera from the ratsare collected during pre-determined time intervals and analyzed forcytokines panel and analyzed for cytokines panel and assay forcholesterol levels as described herein.

B. Non-Human Primates (NHP)

LDLR modified mRNA formulated in 5% sucrose, saline or a lipidnanoparticle is administered to non-human primates intramuscularly orintravenously. The injection contains a dose of LDLR mRNA of between0.005-0.5 mg/kg (e.g. 0.005 mg/kg, 0.010 mg/kg, 0.015 mg/kg, 0.020mg/kg, 0.030 mg/kg, 0.040 mg/kg, 0.050 mg/kg, 0.1 mg/kg, 0.2 mg/kg, 0.3mg/kg, 0.4 mg/kg and 0.5 mg/kg). The LDLR protein expression and druglevel changes in mRNA transcript are measured by western blot analysisusing an anti-LDLR antibody and the level of modified LDLR mRNA in themuscle tissues are analyzed by real time RT-PCR. Sera from the non-humanprimates are collected at predetermined intervals after injection andanalyzed for cytokines panel and assay for cholesterol levels asdescribed herein.

Example 42. Repeat Dose Administration Studies of UGT1A1 Modified mRNAin Mammals

A. Rodents

Studies utilizing multiple doses are designed and performed using ratsand/or mice (e.g. LDLR−/− mice). The rodents are injectedintramuscularly or intravenously more than once (e.g., daily, twice aweek, every 5 days, weekly, every 10 days, bi-weekly) over a period of 4weeks with 0.5 mg/kg, 0.05 mg/kg, 0.005 mg/kg or 0.0005 mg/kg ofmodified LDLR mRNA encoding human or rat LDLR. The LDLR mRNA isformulated in either 5% sucrose, saline or a lipid nanoparticle.

The LDLR protein expression and changes in mRNA transcript is measuredin the cell lysates by western blot analysis using an anti-LDLR antibodyand the drug level and transcript of LDLR in tissues is analyzed by realtime RT-PCR. Sera from the rats are collected during pre-determined timeintervals and analyzed for cytokines panel and assay for cholesterollevels as described herein.

B. Non-Human Primates (NHP)

LDLR modified mRNA formulated in 5% sucrose, saline or a lipidnanoparticle is administered to non-human primates intramuscularly orintravenously more than once (e.g., daily, twice a week, every 5 days,weekly, every 10 days, bi-weekly) over a period of 4 weeks. Theinjection contains a dose of LDLR mRNA of between 0.005-0.5 mg/kg (e.g.0.005 mg/kg, 0.010 mg/kg, 0.015 mg/kg, 0.020 mg/kg, 0.030 mg/kg, 0.040mg/kg, 0.050 mg/kg, 0.1 mg/kg, 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg and 0.5mg/kg).

The non-human primates are weighed prior to the start of the study andweighed at day 8, day 15 and at the end of the study. The LDLR proteinexpression and drug level changes in mRNA transcript are measured bywestern blot analysis using an anti-LDLR antibody and the level ofmodified LDLR mRNA in the muscle tissues are analyzed by real timeRT-PCR. Sera from the non-human primates are collected at predeterminedintervals after injection and analyzed for cytokines panel and assay forcholesterol levels as described herein.

Example 43. Microphysiological Systems

The modified mRNA encoding LDLR and its variants described herein areformulated using one of the methods described herein such as in buffer,lipid nanoparticles and PLGA. These formulations are then administeredto or contacted with microphysiological systems created from organ chipsas described in International Publication Nos. WO2013086502,WO2013086486 and WO2013086505, the contents of each of which are hereinincorporated by reference in its entirety.

Example 44. LDLR Mutations

In one embodiment, the polynucleotides described herein encode at leastone LDLR protein which is deficient is binding to PCSK9. As anon-limiting example, the LDLR protein may comprise at least onemutation to be PCSK9 binding deficient as described herein.

In one embodiment, the polynucleotides described herein may be deficientin binding to disable homolog 2, mitogen-responsive phosphoprotein(DAB2). While not wishing to be bound by theory, the DAB2binding-deficient LDLR may limit the internalization of LDLR through theDAB2 pathway and thus reducing LDLR uptake.

In one embodiment, the NPXY motif of LDLR may be modified in order toalter the signal for rapid endocytosis through coated pits of LDLR. TheNPXY motif may comprise at least one mutation, at least two mutations,at least three mutations, at least four mutations or more than fourmutations. As a non-limiting example, the NPXY motif may comprise aminoacid 822 through amino acid 829 of a LDLR sequence. As anothernon-limiting example, the NPXY motif may comprise the sequence NFDNPVYQ(SEQ ID NO: 62).

In one embodiment, the LDLR sequence does not comprise a mutation in theNPXY motif. In another embodiment, the LDLR sequence may comprise amutation but the mutation may not be at position 822, 826, 827 or 828 ofLDLR where amino acid 822 through amino acid 829 of LDLR is shown in(SEQ ID NO: 62).

In another embodiment, the NPXY motif of LDLR may be modified to reducethe binding of Sorting Nexin 17 (SNX17) to the NPXY motif of LDLR. Thereduction of binding of SNX17 to the NPXY motif of LDLR may be used toregulate the endosomal recycling of receptors.

In one embodiment, the PX domain (PI3P binding) of SNX17 may comprise atleast one mutation. The at least one mutation may alter the ability ofSNX17 to bind to the NPXY motif of LDLR and thus regulate the endosomalrecycling of receptors.

In one embodiment, the FERM-like domain of SNX17 may comprise at leastone mutation. The at least one mutation may alter the ability of SNX17to bind to the NPXY motif of LDLR and thus regulate the endosomalrecycling of receptors.

In one embodiment, the Ras-association domain of SNX17 may comprise atleast one mutation. The at least one mutation may alter the ability ofSNX17 to bind to the NPXY motif of LDLR and thus regulate the endosomalrecycling of receptors.

In one embodiment, a LDLR sequence described herein may comprise atleast one amino acid which has been phosphorylated. As a non-limitingexample, at least one amino acid in the sequence NQDGYSYPSR (SEQ ID NO:63) may be phosphorylated. As a non-limiting example, the two tyrosines(Ys) in SEQ ID NO: 63 of LDLR may be phosphorylated. As anothernon-limiting example, at least one tyrosine (Y) in the LDLR sequencedescribed herein may be phosphorylated. As yet another non-limitingexample, tyrosine at position 845 and tyrosine at position 847 of LDLRdescribed herein are phosphorylated.

In one embodiment, a LDLR sequence described herein may comprise atleast one amino acid which has been phosphorylated but tyrosine atposition 828 is not phosphorylated.

In another embodiment, a LDLR sequences described herein may comprise atleast one amino acid which has been phosphorylated, wherein at least oneof the amino acids is tyrosine at position 828.

In one embodiment, the LDLR sequence described herein may comprise atleast one amino acid mutation in the C-terminal sequence LEDDVA (SEQ IDNO: 64). As a non-limiting example, SEQ ID NO: 64 may be amino acid 855through amino acid 860 of the LDLR sequence.

In one embodiment, the LDLR sequences may comprise at least one mutationat an N-linked glycosylation site of the LDLR sequence. As anon-limiting example, at least one mutation may be located at amino acid97, 156, 272, 515 and/or 657.

In another embodiment, the LDLR sequences may comprise at least onemutation at an O-linked glycosylation site of the LDLR sequence. As anon-limiting example, at least one mutation may be located at aminoacids 721-768.

In yet another embodiment, the LDLR sequences may comprise at least onemutation at an N-linked glycosylation site ant at least one mutation atan O-linked glycosylation site.

In one embodiment, the polynucleotides described herein may be deficientin binding to low density lipoprotein receptor adaptor protein 1(LDLRAP1). While not wishing to be bound by theory, the LDLRAP1binding-deficient LDLR may limit the binding and internalization of LDLRand thus reducing LDLR uptake.

In one embodiment, the ecto-domains of LDLR sequences and constructsdescribed herein may be fused with cytoplasmic domains. As anon-limiting example, LDLR ecto-domain may be fused with folate receptorTM-cytoplasmic domain. As another non-limiting example, LDLR ecto-domainmay be fused with GPI-linked receptor TM-cytoplasmic domain.

Other Embodiments

It is to be understood that the words which have been used are words ofdescription rather than limitation, and that changes may be made withinthe purview of the appended claims without departing from the true scopeand spirit of the invention in its broader aspects.

While the present invention has been described at some length and withsome particularity with respect to the several described embodiments, itis not intended that it should be limited to any such particulars orembodiments or any particular embodiment, but it is to be construed withreferences to the appended claims so as to provide the broadest possibleinterpretation of such claims in view of the prior art and, therefore,to effectively encompass the intended scope of the invention.

All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. In addition, section headings, the materials, methods, andexamples are illustrative only and not intended to be limiting.

1. A composition comprising a synthetic polynucleotide encoding PCSK9negative a low density lipoprotein receptor (LDLR) having one or moreamino acids substituted such that the LDLR mutant is deficient inproprotein convertase subtilisin/kexin type 9 (PCSK9) binding, in anacceptable diluent or carrier.
 2. (canceled)
 3. The composition of claim1, wherein the synthetic polynucleotide comprises: (a) a first region oflinked nucleosides, said first region encoding at least one cholesterolregulating polypeptide; (b) a first flanking region located at the 5′terminus of said first region comprising; (i) a sequence of linkednucleosides selected from the group consisting of the native 5′untranslated region (UTR), SEQ ID NO: 1 and functional variants thereof;(c) a second flanking region located at the 3′ terminus of said firstregion comprising; (i′) a sequence of linked nucleosides selected fromthe group consisting of the native 3′ UTR, any of the forgoingcomprising one or more microRNA or microRNA binding site or microRNAseeds and functional variants or combinations thereof; and (ii′) a 3′tailing sequence of linked nucleosides; wherein the first region oflinked nucleosides comprises at least a first modified nucleoside. 4-6.(canceled)
 7. A method of treating a disease or disorder in a subject inneed thereof comprising administering to said subject the composition ofclaim
 1. 8-16. (canceled)
 17. A method of modulating cholesterol levelsin plasma of a subject comprising contacting said subject with thecomposition of claim
 1. 18. (canceled)
 19. A polynucleotide comprisingan mRNA encoding a LDLR mutant having one or more amino acidssubstituted such that the LDLR mutant is deficient in PCSK9 binding,wherein the one or more amino acids correspond to amino acid 316 of SEQID NO: 19, amino acid 317 of SEQ ID NO:19, amino acid 331 of SEQ IDNO:19, amino acid 336 of SEQ ID NO:19, amino acid 339 of SEQ ID NO:19,or any combination thereof.
 20. The polynucleotide of claim 19, whereinthe LDLR mutant has two or more amino acids substituted such that theLDLR mutant is deficient in PCSK9 binding, wherein the two or moresubstituted amino acids correspond to any two or more of amino acid 316of SEQ ID NO:19, amino acid 317 of SEQ ID NO:19, amino acid 331 of SEQID NO:19, amino acid 336 of SEQ ID NO:19, and amino acid 339 of SEQ IDNO:19.
 21. The polynucleotide of claim 19, wherein the substituted aminoacids comprise one or more of N316A, E317A, D331A, Y336A, L339D, D331E,or any combination thereof.
 22. The polynucleotide of claim 19, whereinthe half-life of the LDLR mutant encoded by the polynucleotide is longerthan the half-life of wild-type LDLR consisting of the sequence setforth as SEQ ID NO:
 19. 23. The polynucleotide of claim 19 whichcomprises: a first region of linked nucleosides encoding the LDLRmutant; (ii) a first flanking region located at the 5′ terminus of saidfirst region comprising a sequence of linked nucleosides comprising a 5′untranslated region (UTR); (iii) a second flanking region located at the3′ terminus of said first region comprising a sequence of linkednucleosides comprising a 3′ UTR; and (iv) a 3′ tailing sequence oflinked nucleosides.
 24. A composition comprising the polynucleotide ofclaim 19 and an excipient.
 25. The composition of claim 24, wherein thepolynucleotide is formulated in a lipid nanoparticle.
 26. A method oftreating or reducing the incidence of a disease or disorder in a humansubject in need thereof comprising administering to the subject thecomposition of claim
 24. 27. The method of claim 26, wherein the diseaseor disorder is selected from the group consisting of fatty liverdisease, hepatocellular carcinoma, NASH, steatosis, familialhypercholesterolemia (FH), hypercholesterolemia, and aberrantlipoprotein profile.
 28. The method of claim 26, wherein theadministration lowers the cholesterol levels in the plasma of thesubject.
 29. The method of claim 26, wherein the subject has apolymorphism in CYP7A1.
 30. The method of claim 26, wherein the mRNA isfully modified with 1-methylpseudouridine, pseudouridine, or thecombination thereof.
 31. The method of claim 26, further comprisingadministering to said subject a synthetic polynucleotide encodingCYP7A1.
 32. A method of lowering cholesterol levels and/or low-densitylipoprotein (LDL) levels in plasma of a human subject comprisingadministering to said subject the composition of claim
 24. 33. Themethod of claim 32, wherein the cholesterol levels in plasma are loweredby at least 20% at 24 hours following administration of thepolynucleotide to the subject.
 34. A method of increasing expression ofreceptors that bind LDL on the surface of a cell comprising contactingthe cell with the polynucleotide of claim 19.